Methods and compositions for determining the sequence of nucleic acid molecules

ABSTRACT

Methods and compounds, including compositions therefrom, are provided for determining the sequence of nucleic acid molecules. The methods permit the determination of multiple nucleic acid sequences simultaneously. The compounds are used as tags to generate tagged nucleic acid fragments which are complementary to a selected target nucleic acid molecule. Each tag is correlative with a particular nucleotide and, in a preferred embodiment, is detectable by mass spectrometry. Following separation of the tagged fragments by sequential length, the tags are cleaved from the tagged fragments. In a preferred embodiment, the tags are detected by mass spectrometry and the sequence of the nucleic acid molecule is determined therefrom. The individual steps of the methods can be used in automated format, e.g., by the incorporation into systems.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/786,835, filed Jan. 22, 1997 now abandoned, whichapplication claims the benefit of provisional application No.60/010,462, filed Jan. 23, 1996, both of which are incorporated byreference herein in their entirety.

TECHNICAL FIELD

The present invention relates generally to methods and compositions fordetermining the sequence of nucleic acid molecules, and morespecifically, to methods and compositions which allow the determinationof multiple nucleic acid sequences simultaneously.

BACKGROUND OF THE INVENTION

Deoxyribonucleic acid (DNA) sequencing is one of the basic techniques ofbiology. It is at the heart of molecular biology and plays a rapidlyexpanding role in the rest of biology. The Human Genome Project is amulti-national effort to read the entire human genetic code. It is thelargest project ever undertaken in biology, and has already begun tohave a major impact on medicine. The development of cheaper and fastersequencing technology will ensure the success of this project. Indeed, asubstantial effort has been funded by the NIH and DOE branches of theHuman Genome Project to improve sequencing technology, however, withouta substantial impact on current practices (Sulston and Waterston, Nature376:175, 1995).

In the past two decades, determination and analysis of nucleic acidsequence has formed one of the building blocks of biological research.This, along with new investigational tools and methodologies, hasallowed scientists to study genes and gene products in order to betterunderstand the function of these genes, as well as to develop newtherapeutics and diagnostics.

Two different DNA sequencing methodologies that were developed in 1977,are still in wide use today. Briefly, the enzymatic method described bySanger (Proc. Natl. Acad. Sci. (USA) 74:5463, 1977) which utilizesdideoxy-terminators, involves the synthesis of a DNA strand from asingle-stranded template by a DNA polymerase. The Sanger method ofsequencing depends on the fact that that dideoxynucleotides (ddNTPs) areincorporated into the growing strand in the same way as normaldeoxynucleotides (albeit at a lower efficiency). However, ddNTPs differfrom normal deoxynucleotides (dNTPs) in that they lack the 3′-OH groupnecessary for chain elongation. When a ddNTP is incorporated into theDNA chain, the absence of the 3′-hydroxy group prevents the formation ofa new phosphodiester bond and the DNA fragment is terminated with theddNTP complementary to the base in the template DNA. The Maxam andGilbert method (Maxam and Gilbert, Proc. Natl. Acad. Sci. (USA) 74:560,1977) employs a chemical degradation method of the original DNA (in bothcases the DNA must be clonal). Both methods produce populations offragments that begin from a particular point and terminate in every basethat is found in the DNA fragment that is to be sequenced. Thetermination of each fragment is dependent on the location of aparticular base within the original DNA fragment. The DNA fragments areseparated by polyacrylamide gel electrophoresis and the order of the DNAbases (adenine, cytosine, thymine, guanine; also known as A,C,T,G,respectively) is read from a autoradiograph of the gel.

A cumbersome DNA pooling sequencing strategy (Church andKieffer-Higgins, Science 24:185, 1988) is one of the more recentapproaches to DNA sequencing. A pooling sequencing strategy consists ofpooling a number of DNA templates (samples) and processing the samplesas pools. In order to separate the sequence information at the end ofthe processing, the DNA molecules of interest are ligated to a set ofoligonucleotide “tags” at the beginning. The tagged DNA molecules arepooled, amplified and chemically fragmented in 96-well plates. Afterelectrophoresis of the pooled samples, the DNA is transferred to a solidsupport and then hybridized with a sequential series of specific labeledoligonucleotides. These membranes are then probed as many times as thereare tags in the original pool, producing, in each set of probing,autoradiographs similar to those from standard DNA sequencing methods.Thus each reaction and gel yields a quantity of data equivalent to thatobtained from conventional reactions and gels multiplied by the numberof probes used. If alkaline phosphatase is used as the reporter enzyme,1,2-dioxetane substrate can be used which is detected in achemiluminescent assay format. However, this pooling strategy's majordisadvantage is that the sequences can only be read by Southern blottingthe sequencing gel and hybridizing this membrane once for each clone inthe pool.

In addition to advances in sequencing methodologies, advances in speedhave occurred due to the advent of automated DNA sequencing. Briefly,these methods use fluorescent-labeled primers which replace methodswhich employed radiolabeled components. Fluorescent dyes are attachedeither to the sequencing primers or the ddNTP-terminators. Roboticcomponents now utilize polymerase chain reaction (PCR) technology whichhas lead to the development of linear amplification strategies. Currentcommercial sequencing allows all 4 dideoxy-terminator reactions to berun on a single lane. Each dideoxy-terminator reaction is represented bya unique fluorescent primer (one fluorophore for each base type:A,T,C,G). Only one template DNA (i.e., DNA sample) is represented perlane. Current gels permit the simultaneous electrophoresis of up to 64samples in 64 different lanes. Different ddNTP-terminated fragments aredetected by the irradiation of the gel lane by light followed bydetection of emitted light from the fluorophore. Each electrophoresisstep is about 4-6 hours long. Each electrophoresis separation resolvesabout 400-600 nucleotides (nt), therefore, about 6000 nt can besequenced per hour per sequencer.

The use of mass spectrometry for the study of monomeric constituents ofnucleic acids has also been described (Hignite, In BiochemicalApplications of Mass Spectrometry, Waller and Dermer (eds.),Wiley-Interscience, Chapter 16, p. 527, 1972). Briefly, for largeroligomers, significant early success was obtained by plasma desorptionfor protected synthetic oligonucleotides up to 14 bases long, and forunprotected oligos up to 4 bases in length. As with proteins, theapplicability of ESI-MS to oligonucleotides has been demonstrated (Coveyet al., Rapid Comm. in Mass Spec. 2:249-256, 1988). These species areionized in solution, with the charge residing at the acidic bridgingphosphodiester and/ or terminal phosphate moieties, and yield in the gasphase multiple charged molecular anions, in addition to sodium adducts.

Sequencing DNA with <100 bases by the common enzymatic ddNTP techniqueis more complicated than it is for larger DNA templates, so thatchemical degradation is sometimes employed. However, the chemicaldecomposition method requires about 50 pmol of radioactive ³²Pend-labeled material, 6 chemical steps, electrophoretic separation, andfilm exposure. For small oligonucleotides (<14 nts) the combination ofelectrospray ionization (ESI) and Fourier transform (FT) massspectrometry (MS) is far faster and more sensitive. Dissociationproducts of multiply-charged ions measured at high (10⁵) resolving powerrepresent consecutive backbone cleavages providing the full sequence inless than one minute on sub-picomole quantity of sample (Little et al.,J. Am. Chem. Soc. 116:4893, 1994). For molecular weight measurements,ESI/MS has been extended to larger fragments (Potier et al., Nuc. AcidsRes. 22:3895, 1994). ESI/FTMS appears to be a valuable complement toclassical methods for sequencing and pinpoint mutations in nucleotidesas large as 100-mers. Spectral data have recently been obtained loading3×10⁻¹³ mol of a 50-mer using a more sensitive ESI source (Valaskovic,Anal. Chem. 68:259, 1995).

The other approach to DNA sequencing by mass spectrometry is one inwhich DNA is labeled with individual isotopes of an element and the massspectral analysis simply has to distinguish the isotopes after amixtures of sizes of DNA have been separated by electrophoresis. (Theother approach described above utilizes the resolving power of the massspectrometer to both separate and detect the DNA oligonucleotides ofdifferent lengths, a difficult proposition at best.) All of theprocedures described below employ the Sanger procedure to convert asequencing primer to a series of DNA fragments that vary in length byone nucleotide. The enzymatically synthesized DNA molecules each containthe original primer, a replicated sequence of part of the DNA ofinterest, and the dideoxy terminator. That is, a set of DNA molecules isproduced that contain the primer and differ in length by from each otherby one nucleotide residue.

Brennen et al. (Biol. Mass Spec., New York, Elsevier, p. 219, 1990) hasdescribed methods to use the four stable isotopes of sulfur as DNAlabels that enable one to detect DNA fragments that have been separatedby capillary electrophoresis. Using the α-thio analogues of the ddNTPs,a single sulfur isotope is incorporated into each of the DNA fragments.Therefore each of the four types of DNA fragments (ddTTP, ddATP, ddGTP,ddCTP-terminated) can be uniquely labeled according to the terminalnucleotide; for example, ³²S for fragments ending in A, ³³S for G, ³⁴Sfor C, and ³⁶S for T, and mixed together for electrophoresis column,fractions of a few picoliters are obtained by a modified ink-jet printerhead, and then subjected to complete combustion in a firnace. Thisprocess oxidizes the thiophosphates of the labeled DNA to SO₂, which issubjected to analysis in a quadrupole or magnetic sector massspectrometer. The SO₂ mass unit representation is 64 for fragmentsending in A, 65 for G, 66 for C, and 68 for T. Maintenance of theresolution of the DNA fragments as they emerge from the column dependson taking sufficiently small fractions. Because the mass spectrometer iscoupled directly to the capillary gel column, the rate of analysis isdetermined by the rate of electrophoresis. This process is unfortunatelyexpensive, liberates radioactive gas and has not been commercialized.Two other basic constraints also operate on this approach: (a) No othercomponents with mass of 64, 65, 66, or 68 (isobaric contaminants) can betolerated and (b) the % natural abundances of the sulfur isotopes (³²Sis 95.0, ³³S is 0.75, ³⁴S is 4.2, and ³⁶S is 0.11) govern thesensitivity and cost. Since ³²S is 95% naturally abundant, the otherisotopes must be enriched to >99% to eliminate contaminating ³²S.Isotopes that are <1% abundant are quite expensive to obtain at 99%enrichment; even when ³⁶S is purified 100-fold it contains as much ormore ³⁴S as it does ³⁶S.

Gilbert has described an automated DNA sequencer (EPA, 92108678.2) thatconsists of an oligomer synthesizer, an array on a membrane, a detectorwhich detects hybridization and a central computer. The synthesizersynthesizes and labels multiple oligomers of arbitrary predictedsequence. The oligomers are used to probe immobilized DNA on membranes.The detector identifies hybridization patterns and then sends thosepatterns to a central computer which constructs a sequence and thenpredicts the sequence of the next round of synthesis of oligomers.Through an iterative process, a DNA sequence can be obtained in anautomated fashion.

Brennen has described a method for sequencing nucleic acids based onligation of oligomers (U.S. Pat. No. 5,403,708). Methods andcompositions are described for forming ligation product hybridized to anucleic acid template. A primer is hybridized to a DNA template and thena pool of random extension oligonucleotides is also hybridized to theprimed template in the presence ligase(s). The ligase enzyme covalentlyligates the hybridized oligomers to the primer. Modifications permit thedetermination of the nucleotide sequence of one or more members of afirst set of target nucleotide residues in the nucleic acid templatethat are spaced at intervals of N nucleotides. In this method, thelabeled ligated product is formed wherein the position and type of labelincorporated into the ligation product provides information concerningthe nucleotide residue in the nucleic acid template with which thelabeled nucleotide residue is base paired.

Koster has described an method for sequencing DNA by mass spectrometryafter degradation of DNA by an exonuclease (PCT/US94/02938). The methoddescribed is simple in that DNA sequence is directly determined (theSanger reaction is not used). DNA is cloned into standard vectors, the5′ end is immobilized and the strands are then sequentially degraded atthe 3′ end via an exonuclease and the enzymatic product (nucleotides)are detected by mass spectrometry.

Weiss et al. have described an automated hybridization/imaging devicefor fluorescent multiplex DNA sequencing (PCT/US94/11918). The method isbased on the concept of hybridizing enzyme-linked probes to a membranecontaining size separated DNA fragments arising from a typical Sangerreaction.

The demand for sequencing information is larger than can be supplied bythe currently existing sequencing machines, such as the ABI377 and thePharmacia ALF. One of the principal limitations of the currenttechnology is the small number of tags which can be resolved using thecurrent tagging system. The Church pooling system discussed above usesmore tags, but the use and detection of these tags is laborious.

The present invention discloses novel compositions and methods which maybe utilized to sequence nucleic acid molecules with greatly increasedspeed and sensitivity than the methods described above, and furtherprovides other related advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides methods, compounds,compositions, kits and systems for determining the sequence of nucleicacid molecules. Within one aspect of the invention, methods are providedfor determining the sequence of a nucleic acid molecule. The methodsincludes the steps: (a) generating tagged nucleic acid fragments whichare complementary to a selected target nucleic acid molecule, wherein atag is correlative with a particular nucleotide and detectable bynon-fluorescent spectrometry or potentiometry; (b) separating the taggedfragments by sequential length; (c) cleaving the tags from the taggedfragments; and (d) detecting the tags by non-fluorescent spectrometry orpotentiometry, and therefrom determining the sequence of the nucleicacid molecule. In preferred embodiments, the tags are detected by massspectrometry, infrared spectrometry, ultraviolet spectrometry orpotentiostatic amperometry.

In another aspect, the invention provides a compound of the formula:

T^(ms)—L—X

wherein T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; Lis an organic group which allows a T^(ms)-containing moiety to becleaved from the remainder of the compound, wherein theT^(ms)-containing moiety comprises a functional group which supports asingle ionized charge state when the compound is subjected to massspectrometry and is selected from tertiary amine, quaternary amine andorganic acid; X is a fuinctional group selected from hydroxyl, amino,thiol, carboxylic acid, haloalkyl, and derivatives thereof which eitheractivate or inhibit the activity of the group toward coupling with othermoieties, or is a nucleic acid fragment attached to L at other than the3′ end of the nucleic acid fragment; with the provisos that the compoundis not bonded to a solid support through X nor has a mass of less than250 daltons.

In another aspect, the invention provides a composition comprising aplurality of compounds of the formula T^(ms)—L—MOI, wherein, T^(ms) isan organic group detectable by mass spectrometry, comprising carbon, atleast one of hydrogen and fluoride, and optional atoms selected fromoxygen, nitrogen, sulfur, phosphorus and iodine; L is an organic groupwhich allows a T^(ms)-containing moiety to be cleaved from the remainderof the compound, wherein the T^(ms)-containing moiety comprises afunctional group which supports a single ionized charge state when thecompound is subjected to mass spectrometry and is selected from tertiaryamine, quaternary amine and organic acid; MOI is a nucleic acid fragmentwherein L is conjugated to the MOI at a location other than the 3′ endof the MOI; and wherein no two compounds have either the same T^(ms) orthe same MOI.

In another aspect, the invention provides a composition comprising waterand a compound of the formula T^(ms)—L—MOI, wherein, T^(ms) is anorganic group detectable by mass spectrometry, comprising carbon, atleast one of hydrogen and fluoride, and optional atoms selected fromoxygen, nitrogen, sulfur, phosphorus and iodine; L is an organic groupwhich allows a T^(ms)-containing moiety to be cleaved from the remainderof the compound, wherein the T^(ms)-containing moiety comprises afunctional group which supports a single ionized charge state when thecompound is subjected to mass spectrometry and is selected from tertiaryamine, quaternary amine and organic acid; and MOI is a nucleic acidfragment wherein L is conjugated to the MOI at a location other than the3′ end of the MOI.

In another aspect, the invention provides for a composition comprising aplurality of sets of compounds, each set of compounds having the formulaT^(ms)—L—MOI, wherein, T^(ms) is an organic group detectable by massspectrometry, comprising carbon, at least one of hydrogen and fluoride,and optional atoms selected from oxygen, nitrogen, sulfur, phosphorusand iodine; L is an organic group which allows a T^(ms)-containingmoiety to be cleaved from the remainder of the compound, wherein theT^(ms)-containing moiety comprises a functional group which supports asingle ionized charge state when the compound is subjected to massspectrometry and is selected from tertiary amine, quaternary amine andorganic acid; MOI is a nucleic acid fragment wherein L is conjugated tothe MOI at a location other than the 3′ end of the MOI; wherein within aset, all members have the same T^(ms) group, and the MOI fragments havevariable lengths that terminate with the same dideoxynucleotide selectedfrom ddAMP, ddGMP, ddCMP and ddTMP; and wherein between sets, the T^(ms)groups differ by at least 2 amu.

In another aspect, the invention provides for a composition comprising afirst plurality of sets of compounds as described in the precedingparagraph, in combination with a second plurality of sets of compoundshaving the formula T^(ms)—L—MOI, wherein, T^(ms) is an organic groupdetectable by mass spectrometry, comprising carbon, at least one ofhydrogen and fluoride, and optional atoms selected from oxygen,nitrogen, sulfur, phosphorus and iodine; L is an organic group whichallows a T^(ms)-containing moiety to be cleaved from the remainder ofthe compound, wherein the T^(ms)-containing moiety comprises afumctional group which supports a single ionized charge state when thecompound is subjected to mass spectrometry and is selected from tertiaryamine, quaternary amine and organic acid; MOI is a nucleic acid fragmentwherein L is conjugated to the MOI at a location other than the 3′ endof the MOI; and wherein all members within the second plurality have anMOI sequence which terminates with the same dideoxynucleotide selectedfrom ddAMP, ddGMP, ddCMP and ddTMP; with the proviso that thedideoxynucleotide present in the compounds of the first plurality is notthe same dideoxynucleotide present in the compounds of the secondplurality.

In another aspect, the invention provides for a kit for DNA sequencinganalysis. The kit comprises a plurality of container sets, eachcontainer set comprising at least five containers, wherein a firstcontainer contains a vector, a second, third, fourth and fifthcontainers contain compounds of the formula T^(ms)—L—MOI wherein, T^(ms)is an organic group detectable by mass spectrometry, comprising carbon,at least one of hydrogen and fluoride, and optional atoms selected fromoxygen, nitrogen, sulfur, phosphorus and iodine; L is an organic groupwhich allows a T^(ms)-containing moiety to be cleaved from the remainderof the compound, wherein the T^(ms)-containing moiety comprises afunctional group which supports a single ionized charge state when thecompound is subjected to mass spectrometry and is selected from tertiaryamine, quaternary amine and organic acid; and MOI is a nucleic acidfragment wherein L is conjugated to the MOI at a location other than the3′ end of the MOI; such that the MOI for the second, third, fourth andfifth containers is identical and complementary to a portion of thevector within the set of containers, and the T^(ms) group within eachcontainer is different from the other T^(ms) groups in the kit.

In another aspect, the invention provides for systems for determiningthe sequence of a nucleic acid molecule in a sample. In one embodiment,a system comprises a system for determining the sequence of a nucleicacid molecule in a sample, the sample including tagged nucleic acidfragments having nucleic acid fragments and tags attached to the nucleicacid fragments, comprising a separation apparatus that separates taggednucleic acid fragments, a cleavage apparatus that receives separatedtagged cleaves nucleic acid fragments and the tags from the nucleic acidfragments, each tag being correlative with a particular nucleotide ofthe nucleic acid fragment and detectable by electrochemical detection,and an apparatus for electrochemical detection that receives and detectselectrochemical signatures of the tags. In a preferred embodiment, thesystem further includes a data processor that correlates theelectrochemical signature of a tag to a particular nucleotide and to aspecific sample. In another embodiment, a system comprises a system fordetermining the sequence of a nucleic acid molecule in a sample, thesample including tagged nucleic acid fragments having nucleic acidfragments and tags attached to the nucleic acid fragments, comprising aseparation apparatus that separates tagged nucleic acid fragments, acleavage apparatus that receives separated tagged nucleic acid fragmentsand cleaves from the nucleic acid fragments, each tag being correlativewith a particular nucleotide of the nucleic acid fragment and detectableby mass spectrometry, a mass spectrometer that receives the tags anddetects a mass of a tag, and a data processor that correlates the massof a tag to a particular nucleotide and to a specific sample.

Within other embodiments of the invention, 4, 5, 10, 15, 20, 25, 30, 35,40, 50, 60, 70, 80, 90, 100, 200, 250, 300, 350, 400, 450 or greaterthan 500 different and unique tagged molecules may be utilized within agiven reaction simultaneously, wherein each tag is unique for a selectednucleic acid fragment, probe, or first or second member, and may beseparately identified.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are set forth below whichdescribe in more detail certain procedures or compositions (e.g.,plasmids, etc.), and are therefore incorporated by reference in theirentirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the flowchart for the synthesis of pentafluorophenylesters of chemically cleavable mass spectroscopy tags, to liberate tagswith carboxyl amide termini.

FIG. 2 depicts the flowchart for the synthesis of pentafluorophenylesters of chemically cleavable mass spectroscopy tags, to liberate tagswith carboxyl acid termini.

FIGS. 3-6 and 8 depict the flowchart for the synthesis oftetrafluorophenyl esters of a set of 36 photochemically cleavable massspec. tags.

FIG. 7 depicts the flowchart for the synthesis of a set of 36amine-terminated photochemically cleavable mass spectroscopy tags.

FIG. 9 depicts the synthesis of 36 photochemically cleavable massspectroscopy tagged oligonucleotides made from the corresponding set of36 tetrafluorophenyl esters of photochemically cleavable massspectroscopy tag acids.

FIG. 10 depicts the synthesis of 36 photochemically cleavable massspectroscopy tagged oligonucleotides made from the corresponding set of36 amine-terminated photochemically cleavable mass spectroscopy tags.

FIG. 11 illustrates the simultaneous detection of multiple tags by massspectrometry.

FIG. 12 shows the mass spectrogram of the alpha-cyano matrix alone.

FIG. 13 depicts a modularly-constructed tagged nucleic acid fragment.

FIGS. 14A-14I show the separation of DNA fragments by HPLC using avariety of different buffer solutions.

FIG. 15 is a schematic representation of a DNA sequencing system inaccordance with an exemplary embodiment of the present invention.

FIG. 16 is a schematic representation of a DNA sequencing system inaccordance with an alternate embodiment of the present invention.

FIGS. 17A and 17B illustrate the preparation of a cleavable tag of thepresent invention.

FIGS. 18A and 18B illustrate the preparation of a cleavable tag of thepresent invention.

FIG. 19 illustrates the preparation of an intermediate compound usefulin the preparation of a cleavable tag of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Briefly stated, in one aspect the present invention provides compoundswherein a molecule of interest, or precursor thereto, is linked via alabile bond (or labile bonds) to a tag. Thus, compounds of the inventionmay be viewed as having the general formula:

T—L—X

wherein T is the tag component, L is the linker component that eitheris, or contains, a labile bond, and X is either the molecule of interest(MOI) component or a fumctional group component (L_(h)) through whichthe MOI may be joined to T—L. Compounds of the invention may thereforebe represented by the more specific general formulas:

T—L—MOI

and

T—L—L_(h)

For reasons described in detail below, sets of T—L—MOI compounds may bepurposely subjected to conditions that cause the labile bond(s) tobreak, thus releasing a tag moiety from the remainder of the compound.The tag moiety is then characterized by one or more analyticaltechniques, to thereby provide direct information about the structure ofthe tag moiety, and (most importantly) indirect information about theidentity of the corresponding MOI.

As a simple illustrative example of a representative compound of theinvention wherein L is a direct bond, reference is made to the followingstructure (i):

In structure (i), T is a nitrogen-containing polycyclic aromatic moietybonded to a carbonyl group, X is a MOI (and specifically a nucleic acidfragment terminating in an amine group), and L is the bond which formsan amide group. The amide bond is labile relative to the bonds in Tbecause, as recognized in the art, an amide bond may be chemicallycleaved (broken) by acid or base conditions which leave the bonds withinthe tag component unchanged. Thus, a tag moiety (i.e., the cleavageproduct that contains T) may be released as shown below:

However, the linker L may be more than merely a direct bond, as shown inthe following illustrative example, where reference is made to anotherrepresentative compound of the invention having the structure (ii) shownbelow:

It is well-known that compounds having an ortho-nitrobenzylamine moiety(see boxed atoms within structure (ii)) are photolytically unstable, inthat exposure of such compounds to actinic radiation of a specifiedwavelength will cause selective cleavage of the benzylamine bond (seebond denoted with heavy line in structure (ii)). Thus, structure (ii)has the same T and MOI groups as structure (i), however the linker groupcontains multiple atoms and bonds within which there is a particularlylabile bond. Photolysis of structure (ii) thus releases a tag moiety(T-containing moiety) from the remainder of the compound, as shownbelow.

The invention thus provides compounds which, upon exposure toappropriate cleavage conditions, undergo a cleavage reaction so as torelease a tag moiety from the remainder of the compound. Compounds ofthe invention may be described in terms of the tag moiety, the MOI (orprecursor thereto, L_(h)), and the labile bond(s) which join the twogroups together. Alternatively, the compounds of the invention may bedescribed in terms of the components from which they are formed. Thus,the compounds may be described as the reaction product of a tagreactant, a linker reactant and a MOI reactant, as follows.

The tag reactant consists of a chemical handle (T_(h)) and a variablecomponent (T_(vc)), so that the tag reactant is seen to have the generalstructure:

T_(vc)—T_(h)

To illustrate this nomenclature, reference may be made to structure(iii), which shows a tag reactant that may be used to prepare thecompound of structure (ii). The tag reactant having structure (iii)contains a tag variable component and a tag handle, as shown below:

In structure (iii), the tag handle (—C(═O)—A) simply provides an avenuefor reacting the tag reactant with the linker reactant to form a T—Lmoiety. The group “A” in structure (iii) indicates that the carboxylgroup is in a chemically active state, so it is ready for coupling withother handles. “A” may be, for example, a hydroxyl group orpentafluorophenoxy, among many other possibilities. The inventionprovides for a large number of possible tag handles which may be bondedto a tag variable component, as discussed in detail below. The tagvariable component is thus a part of “T” in the formula T—L—X, and willalso be part of the tag moiety that forms from the reaction that cleavesL.

As also discussed in detail below, the tag variable component isso-named because, in preparing sets of compounds according to theinvention, it is desired that members of a set have unique variablecomponents, so that the individual members may be distinguished from oneanother by an analytical technique. As one example, the tag variablecomponent of structure (iii) may be one member of the following set,where members of the set may be distinguished by their UV or massspectra:

Likewise, the linker reactant may be described in terms of its chemicalhandles (there are necessarily at least two, each of which may bedesignated as L_(h)) which flank a linker labile component, where thelinker labile component consists of the required labile moiety (L²) andoptional labile moieties (L¹ and L³), where the optional labile moietieseffectively serve to separate L² from the handles L_(h), and therequired labile moiety serves to provide a labile bond within the linkerlabile component. Thus, the linker reactant may be seen to have thegeneral formula:

L_(h)—L¹—L²—L³—L_(h)

The nomenclature used to describe the linker reactant may be illustratedin view of structure (iv), which again draws from the compound ofstructure (ii):

As structure (iv) illustrates, atoms may serve in more than onefunctional role. Thus, in structure (iv), the benzyl nitrogen functionsas a chemical handle in allowing the linker reactant to join to the tagreactant via an amide-forming reaction, and subsequently also serves asa necessary part of the structure of the labile moiety L² in that thebenzylic carbon-nitrogen bond is particularly susceptible to photolyticcleavage. Structure (iv) also illustrates that a linker reactant mayhave an L³ group (in this case, a methylene group), although not have anL¹ group. Likewise, linker reactants may have an L¹ group but not an L³group, or may have L¹ and L³ groups, or may have neither of L¹ nor L³groups. In structure (iv), the presence of the group “P” next to thecarbonyl group indicates that the carbonyl group is protected fromreaction. Given this configuration, the activated carboxyl group of thetag reactant (iii) may cleanly react with the amine group of the linkerreactant (iv) to form an amide bond and give a compound of the formulaT—L—L_(h).

The MOI reactant is a suitably reactive form of a molecule of interest.Where the molecule of interest is a nucleic acid fragment, a suitableMOI reactant is a nucleic acid fragment bonded through its 5′ hydroxylgroup to a phosphodiester group and then to an alkylene chain thatterminates in an amino group. This amino group may then react with thecarbonyl group of structure (iv), (after, of course, deprotecting thecarbonyl group, and preferably after subsequently activating thecarbonyl group toward reaction with the amine group) to thereby join theMOI to the linker.

When viewed in a chronological order, the invention is seen to take atag reactant (having a chemical tag handle and a tag variablecomponent), a linker reactant (having two chemical linker handles, arequired labile moiety and 0-2 optional labile moieties) and a MOIreactant (having a molecule of interest component and a chemicalmolecule of interest handle) to form T—L—MOI. Thus, to form T—L—MOI,either the tag reactant and the linker reactant are first reactedtogether to provide T—L—L_(h), and then the MOI reactant is reacted withT—L—L_(h) so as to provide T—L—MOI, or else (less preferably) the linkerreactant and the MOI reactant are reacted together first to provideL_(h)—L—MOI, and then L_(h)—L—MOI is reacted with the tag reactant toprovide T—L—MOI. For purposes of convenience, compounds having theformula T—L—MOI will be described in terms of the tag reactant, thelinker reactant and the MOI reactant which may be used to form suchcompounds. Of course, the same compounds of formula T—L—MOI could beprepared by other (typically, more laborious) methods, and still fallwithin the scope of the inventive T—L—MOI compounds.

In any event, the invention provides that a T—L—MOI compound besubjected to cleavage conditions, such that a tag moiety is releasedfrom the remainder of the compound. The tag moiety will comprise atleast the tag variable component, and will typically additionallycomprise some or all of the atoms from the tag handle, some or all ofthe atoms from the linker handle that was used to join the tag reactantto the linker reactant, the optional labile moiety L¹ if this group waspresent in T—L—MOI, and will perhaps contain some part of the requiredlabile moiety L² depending on the precise structure of L² and the natureof the cleavage chemistry. For convenience, the tag moiety may bereferred to as the T-containing moiety because T will typicallyconstitute the major portion (in terms of mass) of the tag moiety.

Given this introduction to one aspect of the present invention, thevarious components T, L and X will be described in detail. Thisdescription begins with the following definitions of certain terms,which will be used hereinafter in describing T, L and X.

As used herein, the term “nucleic acid fragment” means a molecule whichis complementary to a selected target nucleic acid molecule (i.e.,complementary to all or a portion thereof), and may be derived fromnature or synthetically or recombinantly produced, includingnon-naturally occurring molecules, and may be in double or singlestranded form where appropriate; and includes an oligonucleotide (e.g.,DNA or RNA), a primer, a probe, a nucleic acid analog (e.g., PNA), anoligonucleotide which is extended in a 5′ to 3′ direction by apolymerase, a nucleic acid which is cleaved chemically or enzymatically,a nucleic acid that is terminated with a dideoxy terminator or capped atthe 3′ or 5′ end with a compound that prevents polymerization at the 5′or 3′ end, and combinations thereof. The complementarity of a nucleicacid fragment to a selected target nucleic acid molecule generally meansthe exhibition of at least about 70% specific base pairing throughoutthe length of the fragment. Preferably the nucleic acid fragmentexhibits at least about 80% specific base pairing; and most preferablyat least about 90%. Assays for determining the percent mismatch (andthus the percent specific base pairing) are well known in the art andare based upon the percent mismatch as a function of the Tm whenreferenced to the fully base paired control.

As used herein, the term “alkyl,” alone or in combination, refers to asaturated, straight-chain or branched-chain hydrocarbon radicalcontaining from 1 to 10, preferably from 1 to 6 and more preferably from1 to 4, carbon atoms. Examples of such radicals include, but are notlimited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl, decyl and the like. Theterm “alkylene” refers to a saturated, straight-chain or branched chainhydrocarbon diradical containing from 1 to 10, preferably from 1 to 6and more preferably from 1 to 4, carbon atoms. Examples of suchdiradicals include, but are not limited to, methylene, ethylene(—CH₂—CH₂—), propylene, and the like.

The term “alkenyl,” alone or in combination, refers to a straight-chainor branched-chain hydrocarbon radical having at least one carbon-carbondouble bond in a total of from 2 to 10, preferably from 2 to 6 and morepreferably from 2 to 4, carbon atoms. Examples of such radicals include,but are not limited to, ethenyl, E- and Z-propenyl, isopropenyl, E- andZ-butenyl, E- and Z-isobutenyl, E- and Z-pentenyl, decenyl and the like.The term “alkenylene” refers to a straight-chain or branched-chainhydrocarbon diradical having at least one carbon-carbon double bond in atotal of from 2 to 10, preferably from 2 to 6 and more preferably from 2to 4, carbon atoms. Examples of such diradicals include, but are notlimited to, methylidene (═CH₂), ethylidene (—CH═CH—), propylidene(—CH₂—CH═CH—) and the like.

The term “alkynyl,” alone or in combination, refers to a straight-chainor branched-chain hydrocarbon radical having at least one carbon-carbontriple bond in a total of from 2 to 10, preferably from 2 to 6 and morepreferably from 2 to 4, carbon atoms. Examples of such radicals include,but are not limited to, ethynyl (acetylenyl), propynyl (propargyl),butynyl, hexynyl, decynyl and the like. The term “alkynylene”, alone orin combination, refers to a straight-chain or branched-chain hydrocarbondiradical having at least one carbon-carbon triple bond in a total offrom 2 to 10, preferably from 2 to 6 and more preferably from 2 to 4,carbon atoms. Examples of such radicals include, but are not limited,ethynylene (—C≡C—), propynylene (—CH₂—C≡C—) and the like.

The term “cycloalkyl,” alone or in combination, refers to a saturated,cyclic arrangement of carbon atoms which number from 3 to 8 andpreferably from 3 to 6, carbon atoms. Examples of such cycloalkylradicals include, but are not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl and the like. The term “cycloalkylene” refers toa diradical form of a cycloalkyl.

The term “cycloalkenyl,” alone or in combination, refers to a cycliccarbocycle containing from 4 to 8, preferably 5 or 6, carbon atoms andone or more double bonds. Examples of such cycloalkenyl radicalsinclude, but are not limited to, cyclopentenyl, cyclohexenyl,cyclopentadienyl and the like. The term “cycloalkenylene” refers to adiradical form of a cycloalkenyl.

The term “aryl” refers to a carbocyclic (consisting entirely of carbonand hydrogen) aromatic group selected from the group consisting ofphenyl, naphthyl, indenyl, indanyl, azulenyl, fluorenyl, andanthracenyl; or a heterocyclic aromatic group selected from the groupconsisting of furyl, thienyl, pyridyl, pyrrolyl, oxazolyly, thiazolyl,imidazolyl, pyrazolyl, 2-pyrazolinyl, pyrazolidinyl, isoxazolyl,isothiazolyl, 1,2,3-oxadiazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl,pyridazinyl, pyrimidinyl, pyrazinyl, 1,3,5-triazinyl, 1,3,5-trithianyl,indolizinyl, indolyl, isoindolyl, 3H-indolyl, indolinyl,benzo[b]furanyl, 2,3-dihydrobenzofuranyl, benzo[b]thiophenyl,1H-indazolyl, benzimidazolyl, benzthiazolyl, purinyl, 4H-quinolizinyl,quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl,quinoxalinyl, 1,8-naphthyridinyl, pteridinyl, carbazolyl, acridinyl,phenazinyl, phenothiazinyl, and phenoxazinyl.

“Aryl” groups, as defined in this application may independently containone to four substituents which are independently selected from the groupconsisting of hydrogen, halogen, hydroxyl, amino, nitro,trifluoromethyl, trifluoromethoxy, alkyl, alkenyl, alkynyl, cyano,carboxy, carboalkoxy, 1,2-dioxyethylene, alkoxy, alkenoxy or alkynoxy,alkylamino, alkenylamino, alkynylamino, aliphatic or aromatic acyl,alkoxy-carbonylamino, alkylsulfonylamino, morpholinocarbonylamino,thiomorpholinocarbonylamino, N-alkyl guanidino, aralkylaminosulfonyl;aralkoxyalkyl; N-aralkoxyurea; N-hydroxylurea; N-alkenylurea;N,N-(alkyl, hydroxyl)urea; heterocyclyl; thioaryloxy-substituted aryl;N,N-(aryl, alkyl)hydrazino; Ar′-substituted sulfonylheterocyclyl;aralkyl-substituted heterocyclyl; cycloalkyl and cycloakenyl-substitutedheterocyclyl; cycloalkyl-fused aryl; aryloxy-substituted alkyl;heterocyclylamino; aliphatic or aromatic acylaminocarbonyl; aliphatic oraromatic acyl-substituted alkenyl; Ar′-substituted aminocarbonyloxy;Ar′, Ar′-disubstituted aryl; aliphatic or aromatic acyl-substitutedacyl; cycloalkylcarbonylalkyl; cycloalkyl-substituted amino;aryloxycarbonylalkyl; phosphorodiamidyl acid or ester;

“Ar” is a carbocyclic or heterocyclic aryl group as defined above havingone to three substituents selected from the group consisting ofhydrogen, halogen, hydroxyl, amnino, nitro, trifluoromethyl,trifluoromethoxy, alkyl, alkenyl, alkynyl, 1,2-dioxymethylene,1,2-dioxyethylene, alkoxy, alkenoxy, alkynoxy, alkylamino, alkenylaminoor alkynylamino, alkylcarbonyloxy, aliphatic or aromatic acyl,alkylcarbonylamino, alkoxycarbonylamino, alkylsulfonylamino, N-alkyl orN,N-dialkyl urea.

The term “alkoxy,” alone or in combination, refers to an alkyl etherradical, wherein the term “alkyl” is as defined above. Examples ofsuitable alkyl ether radicals include, but are not limited to, methoxy,ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy,tert-butoxy and the like.

The term “alkenoxy,” alone or in combination, refers to a radical offormula alkenyl-O—, wherein the term “alkenyl” is as defined aboveprovided that the radical is not an enol ether. Examples of suitablealkenoxy radicals include, but are not limited to, allyloxy, E- andZ-3-methyl-2-propenoxy and the like.

The term “alkynyloxy,” alone or in combination, refers to a radical offormula alkynyl-O—, wherein the term “alkynyl” is as defined aboveprovided that the radical is not an ynol ether. Examples of suitablealkynoxy radicals include, but are not limited to, propargyloxy,2-butynyloxy and the like.

The term “thioalkoxy” refers to a thioether radical of formula alkyl-S—,wherein alkyl is as defined above.

The term “alkylamino,” alone or in combination, refers to a mono- ordi-alkyl-substituted amino radical (i.e., a radical of formula alkyl-NH—or (alkyl)₂—N—), wherein the term “alkyl” is as defined above. Examplesof suitable alkylamino radicals include, but are not limited to,methylamino, ethylamino, propylamino, isopropylamino, t-butylamino,N,N-diethylamino and the like.

The term “alkenylamino,” alone or in combination, refers to a radical offormula alkenyl-NH— or (alkenyl)₂N—, wherein the term “alkenyl” is asdefined above, provided that the radical is not an enamine. An exampleof such alkenylamino radicals is the allylamino radical.

The term “alkynylamino,” alone or in combination, refers to a radical offormula alkynyl-NH— or (alkynyl)₂N—, wherein the term “alkynyl” is asdefined above, provided that the radical is not an ynamine. An exampleof such alkynylamino radicals is the propargyl amino radical.

The term “amide” refers to either —N(R¹)—C(═O)— or —C(═O)—N(R¹)— whereR¹ is defined herein to include hydrogen as well as other groups. Theterm “substituted amide” refers to the situation where R¹ is nothydrogen, while the term “unsubstituted amide” refers to the situationwhere R¹ is hydrogen.

The term “aryloxy,” alone or in combination, refers to a radical offormula aryl-O—, wherein aryl is as defined above. Examples of aryloxyradicals include, but are not limited to, phenoxy, naphthoxy, pyridyloxyand the like.

The term “arylamino,” alone or in combination, refers to a radical offormula aryl-NH—, wherein aryl is as defined above. Examples ofarylamino radicals include, but are not limited to, phenylamino(anilido), naphthylamino, 2-, 3- and 4-pyridylamino and the like.

The term “aryl-fused cycloalkyl,” alone or in combination, refers to acycloalkyl radical which shares two adjacent atoms with an aryl radical,wherein the terms “cycloalkyl” and “aryl” are as defined above. Anexample of an aryl-fused cycloalkyl radical is the benzofused cyclobutylradical.

The term “alkylcarbonylamino,” alone or in combination, refers to aradical of formula alkyl-CONH, wherein the term “alkyl” is as definedabove.

The term “alkoxycarbonylamino,” alone or in combination, refers to aradical of formula alkyl-OCONH—, wherein -the term “alkyl” is as definedabove.

The term “alkylsulfonylamino,” alone or in combination, refers to aradical of formula alkyl-SO₂NH—, wherein the term “alkyl” is as definedabove.

The term “arylsulfonylamino,” alone or in combination, refers to aradical of formula aryl-SO₂NH—, wherein the term “aryl” is as definedabove.

The term “N-alkylurea,” alone or in combination, refers to a radical offormula alkyl-NH—CO—NH—, wherein the term “alkyl” is as defined above.

The termr “N-arylurea,” alone or in combination, refers to a radical offormula aryl-NH—CO—NH—, wherein the term “aryl” is as defined above.

The term “halogen” means fluorine, chlorine, bromine and iodine.

The term “hydrocarbon radical” refers to an arrangement of carbon andhydrogen atoms which need only a single hydrogen atom to be anindependent stable molecule. Thus, a hydrocarbon radical has one openvalence site on a carbon atom, through which the hydrocarbon radical maybe bonded to other atom(s). Alkyl, alkenyl, cycloalkyl, etc. areexamples of hydrocarbon radicals.

The term “hydrocarbon diradical” refers to an arrangement of carbon andhydrogen atoms which need two hydrogen atoms in order to be anindependent stable molecule. Thus, a hydrocarbon radical has two openvalence sites on one or two carbon atoms, through which the hydrocarbonradical may be bonded to other atom(s). Alkylene, alkenylene,alkynylene, cycloalkylene, etc. are examples of hydrocarbon diradicals.

The term “hydrocarbyl” refers to any stable arrangement consistingentirely of carbon and hydrogen having a single valence site to which itis bonded to another moiety, and thus includes radicals known as alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl (without heteroatomincorporation into the aryl ring), arylalkyl, alkylaryl and the like.Hydrocarbon radical is another name for hydrocarbyl.

The term “hydrocarbylene” refers to any stable arrangement consistingentirely of carbon and hydrogen having two valence sites to which it isbonded to other moieties, and thus includes alkylene, alkenylene,alkynylene, cycloalkylene, cycloalkenylene, arylene (without heteroatomincorporation into the arylene ring), arylalkylene, alkylarylene and thelike. Hydrocarbon diradical is another name for hydrocarbylene.

The term “hydrocarbyl-O-hydrocarbylene” refers to a hydrocarbyl groupbonded to an oxygen atom, where the oxygen atom is likewise bonded to ahydrocarbylene group at one of the two valence sites at which thehydrocarbylene group is bonded to other moieties. The terms“hydrocarbyl-S-hydrocarbylene”, “hydrocarbyl-NH-hydrocarbylene” and“hydrocarbyl-amide-hydrocarbylene” have equivalent meanings, whereoxygen has been replaced with sulfur, —NH— or an amide group,respectively.

The term N-(hydrocarbyl)hydrocarbylene refers to a hydrocarbylene groupwherein one of the two valence sites is bonded to a nitrogen atom, andthat nitrogen atom is simultaneously bonded to a hydrogen and ahydrocarbyl group. The term N,N-di(hydrocarbyl)hydrocarbylene refers toa hydrocarbylene group wherein one of the two valence sites is bonded toa nitrogen atom, and that nitrogen atom is simultaneously bonded to twohydrocarbyl groups.

The term “hydrocarbylacyl-hydrocarbylene” refers to a hydrocarbyl groupbonded through an acyl (—C(═O)—) group to one of the two valence sitesof a hydrocarbylene group.

The terms “heterocyclylhydrocarbyl” and “heterocylyl” refer to a stable,cyclic arrangement of atoms which include carbon atoms and up to fouratoms (referred to as heteroatoms) selected from oxygen, nitrogen,phosphorus and sulfur. The cyclic arrangement may be in the form of amonocyclic ring of 3-7 atoms, or a bicyclic ring of 8-11 atoms. Therings may be saturated or unsaturated (including aromatic rings), andmay optionally be benzofused. Nitrogen and sulfur atoms in the ring maybe in any oxidized form, including the quatemized form of nitrogen. Aheterocyclylhydrocarbyl may be attached at any endocyclic carbon orheteroatom which results in the creation of a stable structure.Preferred heterocyclylhydrocarbyls include 5-7 membered monocyclicheterocycles containing one or two nitrogen heteroatoms.

A substituted heterocyclylhydrocarbyl refers to aheterocyclylhydrocarbyl as defined above, wherein at least one ring atomthereof is bonded to an indicated substituent which extends off of thering.

In referring to hydrocarbyl and hydrocarbylene groups, the term“derivatives of any of the foregoing wherein one or more hydrogens isreplaced with an equal number of fluorides” refers to molecules thatcontain carbon, hydrogen and fluoride atoms, but no other atoms.

The term “activated ester” is an ester that contains a “leaving group”which is readily displaceable by a nucleophile, such as an amine, analcohol or a thiol nucleophile. Such leaving groups are well known andinclude, without limitation, N-hydroxysuccinimide,N-hydroxybenzotriazole, halogen (halides), alkoxy includingtetrafluorophenolates, thioalkoxy and the like. The term “protectedester” refers to an ester group that is masked or otherwise unreactive.See, e.g., Greene, “Protecting Groups In Organic Synthesis.”

In view of the above definitions, other chemical terms used throughoutthis application can be easily understood by those of skill in the art.Terms may be used alone or in any combination thereof. The preferred andmore preferred chain lengths of the radicals apply to all suchcombinations.

A. Generation of Tagged Nucleic Acid Fragments

As noted above, one aspect of the present invention provides a generalscheme for DNA sequencing which allows the use of more than 16 tags ineach lane; with continuous detection, the tags can be detected and thesequence read as the size separation is occurring, just as withconventional fluorescence-based sequencing. This scheme is applicable toany of the DNA sequencing techniques based on size separation of taggedmolecules. Suitable tags and linkers for use within the presentinvention, as well as methods for sequencing nucleic acids, arediscussed in more detail below.

1. Tags

“Tag”, as used herein, generally refers to a chemical moiety which isused to uniquely identify a “molecule of interest”, and morespecifically refers to the tag variable component as well as whatevermay be bonded most closely to it in any of the tag reactant, tagcomponent and tag moiety.

A tag which is useful in the present invention possesses severalattributes:

1) It is capable of being distinguished from all other tags. Thisdiscrimination from other chemical moieties can be based on thechromatographic behavior of the tag (particularly after the cleavagereaction), its spectroscopic or potentiometric properties, or somecombination thereof. Spectroscopic methods by which tags are usefullydistinguished include mass spectroscopy (MS), infrared (IR), ultraviolet(UV), and fluorescence, where MS, IR and UV are preferred, and MS mostpreferred spectroscopic methods. Potentiometric amperometry is apreferred potentiometric method.

2) The tag is capable of being detected when present at 10⁻²² to 10⁻⁶mole.

3) The tag possesses a chemical handle through which it can be attachedto the MOI which the tag is intended to uniquely identify. Theattachment may be made directly to the MOI, or indirectly through a“linker” group.

4) The tag is chemically stable toward all manipulations to which it issubjected, including attachment and cleavage from the MOI, and anymanipulations of the MOI while the tag is attached to it.

5) The tag does not significantly interfere with the manipulationsperformed on the MOI while the tag is attached to it. For instance, ifthe tag is attached to an oligonucleotide, the tag must notsignificantly interfere with any hybridization or enzymatic reactions(e.g., PCR sequencing reactions) performed on the oligonucleotide.Similarly, if the tag is attached to an antibody, it must notsignificantly interfere with antigen recognition by the antibody.

A tag moiety which is intended to be detected by a certain spectroscopicor potentiometric method should possess properties which enhance thesensitivity and specificity of detection by that method. Typically, thetag moiety will have those properties because they have been designedinto the tag variable component, which will typically constitute themajor portion of the tag moiety. In the following discussion, the use ofthe word “tag” typically refers to the tag moiety (i.e., the cleavageproduct that contains the tag variable component), however can also beconsidered to refer to the tag variable component itself because that isthe portion of the tag moiety which is typically responsible forproviding the uniquely detectable properties. In compounds of theformula T—L—X, the “T” portion will contain the tag variable component.Where the tag variable component has been designed to be characterizedby, e.g., mass spectrometry, the “T” portion of T—L—X may be referred toas T^(ms). Likewise, the cleavage product from T—L—X that contains T maybe referred to as the T^(ms)-containing moiety. The followingspectroscopic and potentiometric methods may be used to characterizeT^(ms)-containing moieties. a. Characteristics of MS Tags

Where a tag is analyzable by mass spectrometry (i.e., is a MS-readabletag, also referred to herein as a MS tag or “T^(ms)-containing moiety”),the essential feature of the tag is that it is able to be ionized. It isthus a preferred element in the design of MS-readable tags toincorporate therein a chemical functionality which can carry a positiveor negative charge under conditions of ionization in the MS. Thisfeature confers improved efficiency of ion formation and greater overallsensitivity of detection, particularly in electrospray ionization. Thechemical functionality that supports an ionized charge may derive fromT^(ms) or L or both. Factors that can increase the relative sensitivityof an analyte being detected by mass spectrometry are discussed in,e.g., Sunner, J., et al., Anal. Chem. 60:1300-1307 (1988).

A preferred functionality to facilitate the carrying of a negativecharge is an organic acid, such as phenolic hydroxyl, carboxylic acid,phosphonate, phosphate, tetrazole, sulfonyl urea, perfluoro alcohol andsulfonic acid.

Preferred functionality to facilitate the carrying of a positive chargeunder ionization conditions are aliphatic or aromatic amines. Examplesof amine functional groups which give enhanced detectability of MS tagsinclude quaternary amines (i.e., amines that have four bonds, each tocarbon atoms, see Aebersold, U.S. Pat. No. 5,240,859) and tertiaryamines (i.e., amines that have three bonds, each to carbon atoms, whichincludes C═N—C groups such as are present in pyridine, see Hess et al.,Anal. Biochem. 224:373, 1995; Bures et al., Anal. Biochem. 224:364,1995). Hindered tertiary amines are particularly preferred. Tertiary andquaternary amines may be alkyl or aryl. A T^(ms)-containing moiety mustbear at least one ionizable species, but may possess more than oneionizable species. The preferred charge state is a single ionizedspecies per tag. Accordingly, it is preferred that eachT^(ms)-containing moiety (and each tag variable component) contain onlya single hindered amine or organic acid group.

Suitable amine-containing radicals that may form part of theT^(ms)-containing moiety include the following:

The identification of a tag by mass spectrometry is preferably basedupon its molecular mass to charge ratio (m/z). The preferred molecularmass range of MS tags is from about 100 to 2,000 daltons, and preferablythe T^(ms)-containing moiety has a mass of at least about 250 daltons,more preferably at least about 300 daltons, and still more preferably atleast about 350 daltons. It is generally difficult for massspectrometers to distinguish among moieties having parent ions belowabout 200-250 daltons (depending on the precise instrument), and thuspreferred T^(ms)-containing moieties of the invention have masses abovethat range.

As explained above, the T^(ms)-containing moiety may contain atoms otherthan those present in the tag variable component, and indeed other thanpresent in T^(ms) itself. Accordingly, the mass of T^(ms) itself may beless than about 250 daltons, so long as the T^(ms)-containing moiety hasa mass of at least about 250 daltons. Thus, the mass of T^(ms) may rangefrom 15 (i.e., a methyl radical) to about 10,000 daltons, and preferablyranges from 100 to about 5,000 daltons, and more preferably ranges fromabout 200 to about 1,000 daltons.

It is relatively difficult to distinguish tags by mass spectrometry whenthose tags incorporate atoms that have more than one isotope insignificant abundance. Accordingly, preferred T groups which areintended for mass spectroscopic identification (T^(ms) groups), containcarbon, at least one of hydrogen and fluoride, and optional atomsselected from oxygen, nitrogen, sulfur, phosphorus and iodine. Whileother atoms may be present in the T^(ms), their presence can renderanalysis of the mass spectral data somewhat more difficult. Preferably,the T^(ms) groups have only carbon, nitrogen and oxygen atoms, inaddition to hydrogen and/or fluoride.

Fluoride is an optional yet preferred atom to have in a T^(ms) group. Incomparison to hydrogen, fluoride is, of course, much heavier. Thus, thepresence of fluoride atoms rather than hydrogen atoms leads to T^(ms)groups of higher mass, thereby allowing the T^(ms) group to reach andexceed a mass of greater than 250 daltons, which is desirable asexplained above. In addition, the replacement of hydrogen with fluorideconfers greater volatility on the T^(ms)-containing moiety, and greatervolatility of the analyte enhances sensitivity when mass spectrometry isbeing used as the detection method.

The molecular formula of T^(ms) falls within the scope ofC₁₋₅₀₀N₀₋₁₀₀O₀₋₁₀₀S₀₋₁₀P₀₋₁₀H_(α)F_(β)P_(δ) wherein the sum of α, β andδ is sufficient to satisfy the otherwise unsatisfied valencies of the C,N, O, S and P atoms. The designationC₁₋₅₀₀N₀₋₁₀₀O₀₋₁₀₀S₀₋₁₀P₀₋₁₀H_(α)F_(β)I_(δ) means that T^(ms) containsat least one, and may contain any number from 1 to 500 carbon atoms, inaddition to optionally containing as many as 100 nitrogen atoms (“N₀₋”means that T^(ms) need not contain any nitrogen atoms), and as many as100 oxygen atoms, and as many as 10 sulfur atoms and as many as 10phosphorus atoms. The symbols α, β and δ represent the number ofhydrogen, fluoride and iodide atoms in T^(ms), where any two of thesenumbers may be zero, and where the sum of these numbers equals the totalof the otherwise unsatisfied valencies of the C, N, O, S and P atoms.Preferably, T^(ms) has a molecular formula that falls within the scopeof C₁₋₅₀N₀₋₁₀O₀₋₁₀H_(α)F_(β) where the sum of α and β equals the numberof hydrogen and fluoride atoms, respectively, present in the moiety.

b. Characteristics of IR Tags

There are two primary forms of IR detection of organic chemical groups:

Raman scattering IR and absorption IR Raman scattering IR spectra andabsorption IR spectra are complementary spectroscopic methods. Ingeneral, Raman excitation depends on bond polarizability changes whereasIR absorption depends on bond dipole moment changes. Weak IR absorptionlines become strong Raman lines and vice versa. Wavenumber is thecharacteristic unit for IR spectra. There are 3 spectral regions for IRtags which have separate applications: near IR at 12500 to 4000 cm⁻¹,mid IR at 4000 to 600 cm⁻¹, far IR at 600 to 30 cm⁻¹. For the usesdescribed herein where a compound is to serve as a tag to identify anMOI, probe or primer, the mid spectral regions would be preferred. Forexample, the carbonyl stretch (1850 to 1750 cm⁻¹) would be measured forcarboxylic acids, carboxylic esters and amides, and alkyl and arylcarbonates, carbamates and ketones. N—H bending (1750 to 160 cm⁻¹) wouldbe used to identify amines, ammonium ions, and amides. At 1400 to 1250cm⁻¹, R—OH bending is detected as well as the C—N stretch in amides.Aromatic substitution patterns are detected at 900 to 690 cm⁻¹ (C—Hbending, N—H bending for ArNH₂). Saturated C—H, olefins, aromatic rings,double and triple bonds, esters, acetals, ketals, ammonium salts, N—Ocompounds such as oximes, nitro, N-oxides, and nitrates, azo,hydrazones, quinones, carboxylic acids, amides, and lactams all possessvibrational infrared correlation data (see Pretsch et al., Spectral Datafor Structure Determination of Organic Compounds, Springer-Verlag, NewYork, 1989). Preferred compounds would include an aromatic nitrile whichexhibits a very strong nitrile stretching vibration at 2230 to 2210cm⁻¹. Other useful types of compounds are aromatic alkynes which have astrong stretching vibration that gives rise to a sharp absorption bandbetween 2140 and 2100 cm⁻¹. A third compound type is the aromatic azideswhich exhibit an intense absorption band in the 2160 to 2120 cm⁻¹region. Thiocyanates are representative of compounds that have a strongabsorption at 2275 to 2263 cm⁻¹.

c. Characteristics of UV Tags

A compilation of organic chromophore types and their respectiveUV-visible properties is given in Scott (Interpretation of the UVSpectra of Natural Products, Permagon Press, New York, 1962). Achromophore is an atom or group of atoms or electrons that areresponsible for the particular light absorption. Empirical rules existfor the π to π* maxima in conjugated systems (see Pretsch et al.,Spectral Data for Structure Determination of Organic Compounds, p. B65and B70, Springer-Verlag, New York, 1989). Preferred compounds (withconjugated systems) would possess n to π* and π to π* transitions. Suchcompounds are exemplified by Acid Violet 7, Acridine Orange, AcridineYellow G, Brilliant Blue G, Congo Red, Crystal Violet, Malachite Greenoxalate, Metanil Yellow, Methylene Blue, Methyl Orange, Methyl Violet B,Naphtol Green B, Oil Blue N, Oil Red O, 4-phenylazophenol, Safranie O,Solvent Green 3, and Sudan Orange G, all of which are commerciallyavailable (Aldrich, Milwaukee, Wis.). Other suitable compounds arelisted in, e.g., Jane, I., et al., J. Chrom. 323:191-225 (1985).

d. Characteristic of a Fluorescent Tag

Fluorescent probes are identified and quantitated most directly by theirabsorption and fluorescence emission wavelengths and intensities.Emission spectra (fluorescence and phosphorescence) are much moresensitive and permit more specific measurements than absorption spectra.Other photophysical characteristics such as excited-state lifetime andfluorescence anisotropy are less widely used. The most generally usefulintensity parameters are the molar extinction coefficient (ε) forabsorption and the quantum yield (QY) for fluorescence. The value of εis specified at a single wavelength (usually the absorption maximum ofthe probe), whereas QY is a measure of the total photon emission overthe entire fluorescence spectral profile. A narrow optical bandwidth(<20 nm) is usually used for fluorescence excitation (via absorption),whereas the fluorescence detection bandwidth is much more variable,ranging from full spectrum for maximal sensitivity to narrow band (˜20nm) for maximal resolution. Fluorescence intensity per probe molecule isproportional to the product of ε and QY. The range of these parametersamong fluorophores of current practical importance is approximately10,000 to 100,000 cm⁻¹M⁻¹ for ε and 0.1 to 1.0 for QY. Compounds thatcan serve as fluorescent tags are as follows: fluorescein, rhodamine,lambda blue 470, lambda green, lambda red 664, lambda red 665, acridineorange, and propidium iodide, which are commercially available fromLambda Fluorescence Co. (Pleasant Gap, Pa.). Fluorescent compounds suchas nile red, Texas Red, lissamine™, BODIPY™ s are available fromMolecular Probes (Eugene, Oreg.).

e. Characteristics of Potentiometric Tags

The principle of electrochemical detection (ECD) is based on oxidationor reduction of compounds which at certain applied voltages, electronsare either donated or accepted thus producing a current which can bemeasured. When certain compounds are subjected to a potentialdifference, the molecules undergo a molecular rearrangement at theworking electrodes' surface with the loss (oxidation) or gain(reduction) of electrons, such compounds are said to be electronic andundergo electrochemical reactions. EC detectors apply a voltage at anelectrode surface over which the HPLC eluent flows. Electroactivecompounds eluting from the column either donate electrons (oxidize) oracquire electrons (reduce) generating a current peak in real time.Importantly the amount of current generated depends on both theconcentration of the analyte and the voltage applied, with each compoundhaving a specific voltage at which it begins to oxidize or reduce. Thecurrently most popular electrochemical detector is the amperometricdetector in which the potential is kept constant and the currentproduced from the electrochemical reaction is then measured. This typeof spectrometry is currently called “potentiostatic amperometry”.Commercial amperometers are available from ESA, Inc., Chelmford, Mass.

When the efficiency of detection is 100%, the specialized detectors aretermed “coulometric”. Coulometric detectors are sensitive which have anumber of practical advantages with regard to selectivity andsensitivity which make these types of detectors useful in an array. Incoulometric detectors, for a given concentration of analyte, the signalcurrent is plotted as a function of the applied potential (voltage) tothe working electrode. The resultant sigmoidal graph is called thecurrent-voltage curve or hydrodynamic voltammagram (HDV). The HDV allowsthe best choice of applied potential to the working electrode thatpermits one to maximize the observed signal. A major advantage of ECD isits inherent sensitivity with current levels of detection in thesubfemtomole range.

Numerous chemicals and compounds are electrochemically active includingmany biochemicals, pharmaceuticals and pesticides. Chromatographicallycoeluting compounds can be effectively resolved even if their half-wavepotentials (the potential at half signal maximum) differ by only 30-60mV.

Recently developed coulometric sensors provide selectivity,identification and resolution of co-eluting compounds when used asdetectors in liquid chromatography based separations. Therefore, thesearrayed detectors add another set of separations accomplished in thedetector itself. Current instruments possess 16 channels which are inprinciple limited only by the rate at which data can be acquired. Thenumber of compounds which can be resolved on the EC array ischromatographically limited (i.e., plate count limited). However, if twoor more compounds that chromatographically co-elute have a difference inhalf wave potentials of 30-60 mV, the array is able to distinguish thecompounds. The ability of a compound to be electrochemically activerelies on the possession of an EC active group (i.e., —OH, —O, —N, —S).

Compounds which have been successfully detected using coulometricdetectors include 5-hydroxytryptamine, 3-methoxy-4-hydroxyphenyl-glycol,homogentisic acid, dopamine, metanephrine, 3-hydroxykynureninr,acetominophen, 3-hydroxytryptophol, 5-hydroxyindoleacetic acid,octanesulfonic acid, phenol, o-cresol, pyrogallol, 2-nitrophenol,4-nitrophenol, 2,4-dinitrophenol, 4,6-dinitrocresol,3-methyl-2-nitrophenol, 2,4-dichlorophenol, 2,6-dichlorophenol,2,4,5-trichlorophenol, 4-chloro-3-methylphenol, 5-methylphenol,4-methyl-2-nitrophenol, 2-hydroxyaniline, 4-hydroxyaniline,1,2-phenylenediamine, benzocatechin, buturon, chlortholuron, diuron,isoproturon, linuron, methobromuron, metoxuron, monolinuron, monuron,methionine, tryptophan, tyrosine, 4-aminobenzoic acid, 4-hydroxybenzoicacid, 4-hydroxycoumaric acid, 7-methoxycoumarin, apigenin baicalein,caffeic acid, catechin, centaurein, chlorogenic acid, daidzein,datiscetin, diosmetin, epicatechin gallate, epigallo catechin, epigallocatechin gallate, eugenol, eupatorin, ferulic acid, fisetin, galangin,gallic acid, gardenin, genistein, gentisic acid, hesperidin, irigenin,kaemferol, leucoyanidin, luteolin, mangostin, morin, myricetin,naringin, narirutin, pelargondin, peonidin, phloretin, pratensein,protocatechuic acid, rhamnetin, quercetin, sakuranetin, scutellarein,scopoletin, syringaldehyde, syringic acid, tangeritin, troxerutin,umbelliferone, vanillic acid, 1,3-dimethyl tetrahydroisoquinoline,6-hydroxydopamine, r-salsolinol, N-methyl-r-salsolinol,tetrahydroisoquinoline, amitriptyline, apomorphine, capsaicin,chlordiazepoxide, chlorpromazine, daunorubicin, desipramine, doxepin,fluoxetine, flurazepam, imipramine, isoproterenol, methoxamine,morphine, morphine-3-glucuronide, nortriptyline, oxazepam,phenylephrine, trimipramine, ascorbic acid, N-acetyl serotonin,3,4-dihydroxybenzylamine, 3,4-dihydroxymandelic acid (DOMA),3,4-dihydroxyphenylacetic acid (DOPAC), 3,4-dihydroxyphenylalanine(L-DOPA), 3,4-dihydroxyphenylglycol (DHPG), 3-hydroxyanthranilic acid,2-hydroxyphenylacetic acid (2HPAC), 4-hydroxybenzoic acid (4HBAC),5-hydroxyindole-3-acetic acid (5HIAA), 3-hydroxykynurenine,3-hydroxymandelic acid, 3-hydroxy-4-methoxyphenylethylamine,4-hydroxyphenylacetic acid (4HPAC), 4-hydroxyphenyllactic acid (4HPLA),5-hydroxytryptophan (5HTP), 5-hydroxytryptophol (5HTOL),5-hydroxytryptamine (5HT), 5-hydroxytryptamine sulfate,3-methoxy-4-hydroxyphenylglycol (MHPG), 5-methoxytryptamine,5-methoxytryptophan, 5-methoxytryptophol, 3-methoxytyramine (3MT),3-methoxytyrosine (3-OM-DOPA), 5-methylcysteine, 3-methylguanine,bufotenin, dopamine dopamine-3-glucuronide, dopamine-3-sulfate,dopamine-4-sulfate, epinephrine, epinine, folic acid, glutathione(reduced), guanine, guanosine, homogentisic acid (HGA), homovanillicacid (HVA), homovanillyl alcohol (HVOL), homoveratic acid, hva sulfate,hypoxanthine, indole, indole-3-acetic acid, indole-3-lactic acid,kynurenine, melatonin, metanephrine, N-methyltryptamine,N-methyltyramine, N,N-dimethyltryptamine, N,N-dimethyltyramine,norepinephrine, normetanephrine, octopamine, pyridoxal, pyridoxalphosphate, pyridoxamine, synephrine, tryptophol, tryptamine, tyramine,uric acid, vanillylmandelic acid (vma), xanthine and xanthosine. Othersuitable compounds are set forth in, e.g., Jane, I., et al. J. Chrom.323:191-225 (1985) and Musch, G., et al., J. Chrom. 348:97-110 (1985).These compounds can be incorporated into compounds of formula T—L—X bymethods known in the art. For example, compounds having a carboxylicacid group may be reacted with amine, hydroxyl, etc. to form amide,ester and other linkages between T and L.

In addition to the above properties, and regardless of the intendeddetection method, it is preferred that the tag have a modular chemicalstructure. Ihis aids in the construction of large numbers ofstructurally related tags using the techniques of combinatorialchemistry. For example, the T^(ms) group desirably has severalproperties. It desirably contains a functional group which supports asingle ionized charge state when the T^(ms)-containing moiety issubjected to mass spectrometry (more simply referred to as a “mass specsensitivity enhancer” group, or MSSE). Also, it desirably can serve asone member in a family of T^(ms)-containing moieties, where members ofthe family each have a different mass/charge ratio, however haveapproximately the same sensitivity in the mass spectrometer. Thus, themembers of the family desirably have the same MSSE. In order to allowthe creation of families of compounds, it has been found convenient togenerate tag reactants via a modular synthesis scheme, so that the tagcomponents themselves may be viewed as comprising modules.

In a preferred modular approach to the structure of the T^(ms) group,T^(ms) has the formula

 T²—(J—T³—)_(n)—

wherein T² is an organic moiety formed from carbon and one or more ofhydrogen, fluoride, iodide, oxygen, nitrogen, sulfur and phosphorus,having a mass range of 15 to 500 daltons; T³ is an organic moiety formedfrom carbon and one or more of hydrogen, fluoride, iodide, oxygen,nitrogen, sulfur and phosphorus, having a mass range of 50 to 1000daltons; J is a direct bond or a functional group such as amide, ester,amine, sulfide, ether, thioester, disulfide, thioether, urea, thiourea,carbamate, thiocarbamate, Schiff base, reduced Schiff base, imine,oxime, hydrazone, phosphate, phosphonate, phosphoramide, phosphonamide,sulfonate, sulfonamide or carbon-carbon bond; and n is an integerranging from 1 to 50, such that when n is greater than 1, each T³ and Jis independently selected.

The modular structure T²—(J—T³)_(n)— provides a convenient entry tofamilies of T—L—X compounds, where each member of the family has adifferent T group. For instance, when T is T^(ms), and each familymember desirably has the same MSSE, one of the T³ groups can providethat MSSE structure. In order to provide variability between members ofa family in terms of the mass of T^(ms), the T² group may be variedamong family members. For instance, one family member may haveT²=methyl, while another has T²=ethyl, and another has T²=propyl, etc.

In order to provide “gross” or large jumps in mass, a T³ group may bedesigned which adds significant (e.g, one or several hundreds) of massunits to T—L—X. Such a T³ group may be referred to as a molecular weightrange adjuster group (“WRA”). A WRA is quite useful if one is workingwith a single set of T² groups, which will have masses extending over alimited range. A single set of T² groups may be used to create T^(ms)groups having a wide range of mass simply by incorporating one or moreWRA T³ groups into the T^(ms) . Thus, using a simple example, if a setof T² groups affords a mass range of 250-340 daltons for the T^(ms), theaddition of a single WRA, having, as an exemplary number 100 dalton, asa T³ group provides access to the mass range of 350-440 daltons whileusing the same set of T² groups. Similarly, the addition of two 100dalton MWA groups (each as a T³ group) provides access to the mass rangeof 450-540 daltons, where this incremental addition of WRA groups can becontinued to provide access to a very large mass range for the T^(ms)group. Preferred compounds of the formula T²—(J—T³—)_(n)—L—X have theformula R_(VWC)—(R_(WRA))_(W)—R_(MSSE)—L—X where VWC is a “T²” group,and each of the WRA and MSSE groups are “T³” groups. This structure isillustrated in FIG. 13, and represents one modular approach to thepreparation of T^(ms).

In the formula T²—(J—T³—)_(n)—, T² and T³ are preferably selected fromhydrocarbyl, hydrocarbyl-O-hydrocarbylene, hydrocarbyl-S-hydrocarbylene,hydrocarbyl-NH-hydrocarbylene, hydrocarbyl-amide-hydrocarbylene,N-(hydrocarbyl)hydrocarbylene, N,N-di(hydrocarbyl)hydrocarbylene,hydrocarbylacyl-hydrocarbylene, heterocyclylhydrocarbyl wherein theheteroatom(s) are selected from oxygen, nitrogen, sulfur and phosphorus,substituted heterocyclylhydrocarbyl wherein the heteroatom(s) areselected from oxygen, nitrogen, sulfur and phosphorus and thesubstituents are selected from hydrocarbyl,hydrocarbyl-O-hydrocarbylene, hydrocarbyl-NH-hydrocarbylene,hydrocarbyl-S-hydrocarbylene, N-(hydrocarbyl)hydrocarbylene,N,N-di(hydrocarbyl)hydrocarbylene and hydrocarbylacyl-hydrocarbylene. Inaddition, T² and/or T³ may be a derivative of any of the previouslylisted potential T²/T³ groups, such that one or more hydrogens arereplaced fluorides.

Also regarding the formula T²—(J—T³—)_(n)—, a preferred T³ has theformula —G(R²)—, wherein G is C₁₋₆ alkylene chain having a single R²substituent. Thus, if G is ethylene (—CH₂—CH₂—) either one of the twoethylene carbons may have a R² substituent, and R² is selected fromalkyl, alkenyl, alkynyl, cycloalkyl, aryl-fused cycloalkyl,cycloalkenyl, aryl, aralkyl, aryl-substituted alkenyl or alkynyl,cycloalkyl-substituted alkyl, cycloalkenyl-substituted cycloalkyl,biaryl, alkoxy, alkenoxy, alkynoxy, aralkoxy, aryl-substituted alkenoxyor alkynoxy, alkylamino, alkenylamino or alkynylamino, aryl-substitutedalkylamino, aryl-substituted alkenylamino or alkynylamino, aryloxy,arylarnino, N-alkylurea-substituted alkyl, N-arylurea-substituted alkyl,alkylcarbonylamino-substituted alkyl, aminocarbonyl-substituted alkyl,heterocyclyl, heterocyclyl-substituted alkyl, heterocyclyl-substitutedamino, carboxyalkyl substituted aralkyl, oxocarbocyclyl-fused aryl andheterocyclylalkyl; cycloalkenyl, aryl-substituted alkyl and, aralkyl,hydroxy-substituted alkyl, alkoxy-substituted alkyl,aralkoxy-substituted alkyl, alkoxy-substituted alkyl,aralkoxy-substituted alkyl, amino-substituted alkyl, (aryl-substitutedalkyloxycarbonylamino)-substituted alkyl, thiol-substituted alkyl,alkylsulfonyl-substituted alkyl, (hydroxy-substitutedalkylthio)-substituted alkyl, thioalkoxy-substituted alkyl,hydrocarbylacylamino-substituted alkyl,heterocyclylacylamino-substituted alkyl,hydrocarbyl-substituted-heterocyclylacylamino-substituted alkyl,alkylsulfonylamino-substituted alkyl, arylsulfonylamino-substitutedalkyl, morpholino-alkyl, thiomorpholino-alkyl, morpholinocarbonyl-substituted alkyl, thiomorpholinocarbonyl-substituted alkyl,[N-(alkyl, alkenyl or alkynyl)- or N,N-[dialkyl, dialkenyl, dialkynyl or(alkyl, alkenyl)-amino]carbonyl-substituted alkyl,heterocyclylaminocarbonyl, heterocylylalkyleneaminocarbonyl,heterocyclylaminocarbonyl-substituted alkyl,heterocylylalkyleneaminocarbonyl-substituted alkyl,N,N-[dialkyl]alkyleneaminocarbonyl,N,N-[dialkyl]alkyleneaminocarbonyl-substituted alkyl, alkyl-substitutedheterocyclylcarbonyl, alkyl-substituted heterocyclylcarbonyl-alkyl,carboxyl-substituted alkyl, dialkylamino-substituted acylaminoalkyl andamino acid side chains selected from arginine, asparagine, glutamine,S-methyl cysteine, methionine and corresponding sulfoxide and sulfonederivatives thereof, glycine, leucine, isoleucine, allo-isoleucine,tert-leucine, norleucine, phenylalanine, tyrosine, tryptophan, proline,alanine, omithine, histidine, glutamine, valine, threonine, serine,aspartic acid, beta-cyanoalanine, and allothreonine; alynyl andheterocyclylcarbonyl, aminocarbonyl, amido, mono- ordialkylaminocarbonyl, mono- or diarylaminocarbonyl,alkylarylaminocarbonyl, diarylaminocarbonyl, mono- ordiacylaminocarbonyl, aromatic or aliphatic acyl, alkyl optionallysubstituted by substituents selected from amino, carboxy, hydroxy,mercapto, mono- or dialkylamino, mono- or diarylamino, alkylarylamino,diarylamino, mono- or diacylamino, alkoxy, alkenoxy, aryloxy,thioalkoxy, thioalkenoxy, thioalkynoxy, thioaryloxy and heterocyclyl.

A preferred compound of the formula T²—(J—T³—)_(n)—L—X has thestructure:

wherein G is (CH₂)₁₋₆ such that a hydrogen on one and only one of theCH₂ groups represented by a single “G” is replaced with—(CH₂)_(c)-Amide-T⁴; T² and T⁴ are organic moieties of the formulaC₁₋₂₅N₀₋₉O₀₋₉H_(α)F_(β) such that the sum of α and β is sufficient tosatisfy the otherwise unsatisfied valencies of the C, N, and O atoms;amide is

R¹ is hydrogen or C₁₋₁₀ alkyl; c is an integer ranging from 0 to 4; andn is an integer ranging from 1 to 50 such that when n is greater than 1,G, c, Amide, R¹ and T⁴ are independently selected.

In a further preferred embodiment, a compound of the formulaT²—(J—T³—)_(n)—L—X has the structure:

wherein T⁵ is an organic moiety of the formula C₁₋₂₅N_(0-N)₀₋₉O₀₋₉H_(α)F_(β) such that the sum of α and β is sufficient to satisfythe otherwise unsatisfied valencies of the C, N, and O atoms; and T⁵includes a tertiary or quaternary amine or an organic acid; m is aninteger ranging from 0-49, and T², T⁴, R¹, L and X have been previouslydefined.

Another preferred compound having the formula T²—(J—T³—)_(n)—L—X has theparticular structure:

wherein T⁵ is an organic moiety of the formula C₁₋₂₅N₀₋₉O₀₋₉H_(α)F_(β)such that the sum of α and β is sufficient to satisfy the otherwiseunsatisfied valencies of the C, N, and O atoms; and T⁵ includes atertiary or quaternary amine or an organic acid; m is an integer rangingfrom 0-49, and T², T⁴, c, R¹, “Amide”, L and X have been previouslydefined.

In the above structures that have a T⁵ group, -Amide-T⁵ is preferablyone of the following, which are conveniently made by reacting organicacids with free amino groups extending from “G”:

Where the above compounds have a T⁵ group, and the “G” group has a freecarboxyl group (or reactive equivalent thereof), then the following arepreferred -Amide-T⁵ group, which may conveniently be prepared byreacting the appropriate organic amine with a free carboxyl groupextending from a “G” group:

In three preferred embodiments of the invention, T—L—MOI has thestructure:

or the structure:

or the structure:

wherein T² and T⁴ are organic moieties of the formulaC₁₋₂₅N₀₋₉O₀₋₉S₀₋₃P₀₋₃H_(α)F_(β)I_(δ) such that the sum of α, β and δ issufficient to satisfy the otherwise unsatisfied valencies of the C, N,O, S and P atoms; G is (CH₂)₁₋₆ wherein one and only one hydrogen on theCH₂ groups represented by each G is replaced with —(CH₂)_(c)-Amide-T⁴;Amide is

R¹ is hydrogen or C₁₋₁₀ alkyl; c is an integer ranging from 0 to 4;“C₂-C₁₀” represents a hydrocarbylene group having from 2 to 10 carbonatoms, “ODN-3′-OH” represents a nucleic acid fragment having a terminal3′ hydroxyl group (i.e., a nucleic acid fragment joined to (C₁-C₁₀) atother than the 3′ end of the nucleic acid fragment); and n is an integerranging from 1 to 50 such that when n is greater than 1, then G, c,Amide, R¹ and T⁴ are independently selected. Preferably there are notthree heteroatoms bonded to a single carbon atom.

wherein T² and T⁴ are organic moieties of the formulaC₁₋₂₅N₀₋₉O₀₋₉H_(α)F_(β) such that the sum of α and β is sufficient tosatisfy the otherwise unsatisfied valencies of the C, N, and O atoms; Gis (CH₂)₁₋₆ wherein one and only one hydrogen on the CH₂ groupsrepresented by each G is replaced with —(CH₂)_(c)-Amide-T⁴; Amide is

R¹ is hydrogen or C₁₋₁₀ alkyl; c is an integer ranging from 0 to 4;“ODN-3′-OH” represents a nucleic acid fragment having a terminal 3′hydroxyl group; and n is an integer ranging from 1 to 50 such that whenn is greater than 1, G, c, Amide, R¹ and T⁴ are independently selected.

In structures as set forth above that contain a T²—C(═O)—N(R¹)— group,this group may be formed by reacting an amine of the formula HN(R¹)—with an organic acid selected from the following, which are exemplaryonly and do not constitute an exhaustive list of potential organicacids: Formic acid, Acetic acid, Propiolic acid, Propionic acid,Fluoroacetic acid, 2-Butynoic acid, Cyclopropanecarboxylic acid, Butyricacid, Methoxyacetic acid, Difluoroacetic acid, 4-Pentynoic acid,Cyclobutanecarboxylic acid, 3,3-Dimethylacrylic acid, Valeric acid,N,N-Dimethylglycine, N-Formyl-Gly-OH, Ethoxyacetic acid,(Methylthio)acetic acid, Pyrrole-2-carboxylic acid, 3-Furoic acid,Isoxazole-5-carboxylic acid, trans-3-Hexenoic acid, Trifluoroaceticacid, Hexanoic acid, Ac-Gly-OH, 2-Hydroxy-2-methylbutyric acid, Benzoicacid, Nicotinic acid, 2-Pyrazinecarboxylic acid,1-Methyl-2-pyrrolecarboxylic acid, 2-Cyclopentene-1-acetic acid,Cyclopentylacetic acid, (S)-(−)-2-Pyrrolidone-5-carboxylic acid,N-Methyl-L-proline, Heptanoic acid, Ac-b-Ala-OH,2-Ethyl-2-hydroxybutyric acid, 2-(2-Methoxyethoxy)acetic acid, p-Toluicacid, 6-Methylnicotinic acid, 5-Methyl-2-pyrazinecarboxylic acid,2,5-Dimethylpyrrole-3-carboxylic acid, 4-Fluorobenzoic acid,3,5-Dimethylisoxazole-4-carboxylic acid, 3-Cyclopentylpropionic acid,Octanoic acid, N,N-Dimethylsuccinamic acid, Phenylpropiolic acid,Cinnamic acid, 4-Ethylbenzoic acid, p-Anisic acid,1,2,5-Trimethylpyrrole-3-carboxylic acid, 3-Fluoro-4-methylbenzoic acid,Ac-DL-Propargylglycine, 3-(Trifluoromethyl)butyric acid,1-Piperidinepropionic acid, N-Acetylproline, 3,5-Difluorobenzoic acid,Ac-L-Val-OH, Indole-2-carboxylic acid, 2-Benzofurancarboxylic acid,Benzotriazole-5-carboxylic acid, 4-n-Propylbenzoic acid,3-Dimethylaminobenzoic acid, 4-Ethoxybenzoic acid, 4-(Methylthio)benzoicacid, N-(2-Furoyl)glycine, 2-(Methylthio)nicotinic acid,3-Fluoro-4-methoxybenzoic acid, Tfa-Gly-OH, 2-Napthoic acid, Quinaldicacid, Ac-L-Ile-OH, 3-Methylindene-2-carboxylic acid,2-Quinoxalinecarboxylic acid, 1-Methylindole-2-carboxylic acid,2,3,6-Trifluorobenzoic acid, N-Formyl-L-Met-OH,2-[2-(2-Methoxyethoxy)ethoxy]acetic acid, 4-n-Butylbenzoic acid,N-Benzoylglycine, 5-Fluoroindole-2-carboxylic acid, 4-n-Propoxybenzoicacid, 4-Acetyl-3,5-dimethyl-2-pyrrolecarboxylic acid,3,5-Dimethoxybenzoic acid, 2,6-Dimethoxynicotinic acid,Cyclohexanepentanoic acid, 2-Naphthylacetic acid,4-(1H-Pyrrol-1-yl)benzoic acid, Indole-3-propionic acid,m-Trifluoromethylbenzoic acid, 5-Methoxyindole-2-carboxylic acid,4-Pentylbenzoic acid, Bz-b-Ala-OH, 4-Diethylaminobenzoic acid,4-n-Butoxybenzoic acid, 3-Methyl-5-CF3-isoxazole-4-carboxylic acid,(3,4-Dimethoxyphenyl)acetic acid, 4-Biphenylcarboxylic acid,Pivaloyl-Pro-OH, Octanoyl-Gly-OH, (2-Naphthoxy)acetic acid,Indole-3-butyric acid, 4-(Trifluoromethyl)phenylacetic acid,5-Methoxyindole-3-acetic acid, 4-(Trifluoromethoxy)benzoic acid,Ac-L-Phe-OH, 4-Pentyloxybenzoic acid, Z-Gly-OH,4-Carboxy-N-(fur-2-ylmethyl)pyrrolidin-2-one, 3,4-Diethoxybenzoic acid,2,4-Dimethyl-5-CO₂Et-pyrrole-3-carboxylic acid,N-(2-Fluorophenyl)succinamic acid, 3,4,5-Trimethoxybenzoic acid,N-Phenylanthranilic acid, 3-Phenoxybenzoic acid, Nonanoyl-Gly-OH,2-Phenoxypyridine-3-carboxylic acid,2,5-Dimethyl-1-phenylpyrrole-3-carboxylic acid,trans-4-(Trifluoromethyl)cinnamic acid,(5-Methyl-2-phenyloxazol-4-yl)acetic acid, 4-(2-Cyclohexenyloxy)benzoicacid, 5-Methoxy-2-methylindole-3-acetic acid, trans-4-Cotininecarboxylicacid, Bz-5-Aminovaleric acid, 4-Hexyloxybenzoic acid,N-(3-Methoxyphenyl)succinamic acid, Z-Sar-OH,4-(3,4-Dimethoxyphenyl)butyric acid, Ac-o-Fluoro-DL-Phe-OH,N-(4-Fluorophenyl)glutaramic acid, 4′-Ethyl-4-biphenylcarboxylic acid,1,2,3,4-Tetrahydroacridinecarboxylic acid, 3-Phenoxyphenylacetic acid,N-(2,4-Difluorophenyl)succinamic acid, N-Decanoyl-Gly-OH,(+)-6-Methoxy-a-methyl-2-naphthaleneacetic acid,3-(Trifluoromethoxy)cinnamic acid, N-Formyl-DL-Trp-OH,(R)-(+)-a-Methoxy-a-(trifluoromethyl)phenylacetic acid, Bz-DL-Leu-OH,4-(Trifluoromethoxy)phenoxyacetic acid, 4-Heptyloxybenzoic acid,2,3,4-Trimethoxycinnamic acid, 2,6-Dimethoxybenzoyl-Gly-OH,3-(3,4,5-Trimethoxyphenyl)propionic acid,2,3,4,5,6-Pentafluorophenoxyacetic acid,N-(2,4-Difluorophenyl)glutaramic acid, N-Undecanoyl-Gly-OH,2-(4-Fluorobenzoyl)benzoic acid, 5-Trifluoromethoxyindole-2-carboxylicacid, N-(2,4-Difluorophenyl)diglycolamic acid, Ac-L-Trp-OH,Tfa-L-Phenylglycine-OH, 3-Iodobenzoic acid,3-(4-n-Pentylbenzoyl)propionic acid, 2-Phenyl-4-quinolinecarboxylicacid, 4-Octyloxybenzoic acid, Bz-L-Met-OH, 3,4,5-Triethoxybenzoic acid,N-Lauroyl-Gly-OH, 3,5-Bis(trifluoromethyl)benzoic acid,Ac-5-Methyl-DL-Trp-OH, 2-Iodophenylacetic acid, 3-Iodo-4-methylbenzoicacid, 3-(4-n-Hexylbenzoyl)propionic acid, N-Hexanoyl-L-Phe-OH,4-Nonyloxybenzoic acid, 4′-(Trifluoromethyl)-2-biphenylcarboxylic acid,Bz-L-Phe-OH, N-Tridecanoyl-Gly-OH, 3,5-Bis(trifluoromethyl)phenylaceticacid, 3-(4-n-Heptylbenzoyl)propionic acid, N-Hepytanoyl-L-Phe-OH,4-Decyloxybenzoic acid, N-(α,α,α-trifluoro-m-tolyl)anthranilic acid,Niflumic acid, 4-(2-Hydroxyhexafluoroisopropyl)benzoic acid,N-Myristoyl-Gly-OH, 3-(4-n-Octylbenzoyl)propionic acid,N-Octanoyl-L-Phe-OH, 4-Undecyloxybenzoic acid,3-(3,4,5-Trimethoxyphenyl)propionyl-Gly-OH, 8-Iodonaphthoic acid,N-Pentadecanoyl-Gly-OH, 4-Dodecyloxybenzoic acid, N-Palmitoyl-Gly-OH,and N-Stearoyl-Gly-OH. These organic acids are available from one ormore of Advanced ChemTech, Louisville, Ky.; Bachem Bioscience Inc.,Torrance, Calif.; Calbiochem-Novabiochem Corp., San Diego, Calif.;Farchan Laboratories Inc., Gainesville Fla.; Lancaster Synthesis,Windham N.H.; and MayBridge Chemical Company (c/o Ryan Scientific),Columbia, S.C. The catalogs from these companies use the abbreviationswhich are used above to identify the acids.

f. Combinatorial Chemistry as a Means for Preparing Tags

Combinatorial chemistry is a type of synthetic strategy which leads tothe production of large chemical libraries (see, for example, PCTApplication Publication No. WO 94/08051). These combinatorial librariescan be used as tags for the identification of molecules of interest(MOIs). Combinatorial chemistry may be defined as the systematic andrepetitive, covalent connection of a set of different “building blocks”of varying structures to each other to yield a large array of diversemolecular entities. Building blocks can take many forms, both naturallyoccurring and synthetic, such as nucleophiles, electrophiles, dienes,alkylating or acylating agents, diamines, nucleotides, amino acids,sugars, lipids, organic monomers, synthons, and combinations of theabove. Chemical reactions used to connect the building blocks mayinvolve alkylation, acylation, oxidation, reduction, hydrolysis,substitution, elimination, addition, cyclization, condensation, and thelike. This process can produce libraries of compounds which areoligomeric, non-oligomeric, or combinations thereof. If oligomeric, thecompounds can be branched, unbranched, or cyclic. Examples of oligomericstructures which can be prepared by combinatorial methods includeoligopeptides, oligonucleotides, oligosaccharides, polylipids,polyesters, polyamides, polyurethanes, polyureas, polyethers,poly(phosphorus derivatives), e.g., phosphates, phosphonates,phosphoramides, phosphonamides, phosphites, phosphinamides, etc., andpoly(sulfuir derivatives), e.g., sulfones, sulfonates, sulfites,sulfonamides, sulfenamides, etc.

One common type of oligomeric combinatorial library is the peptidecombinatorial library. Recent innovations in peptide chemistry andmolecular biology have enabled libraries consisting of tens to hundredsof millions of different peptide sequences to be prepared and used. Suchlibraries can be divided into three broad categories. One category oflibraries involves the chemical synthesis of soluble non-support-boundpeptide libraries (e.g., Houghten et al., Nature 354:84, 1991). A secondcategory involves the chemical synthesis of support-bound peptidelibraries, presented on solid supports such as plastic pins, resinbeads, or cotton (Geysen et al., Mol. Immunol. 23:709, 1986; Lam et al.,Nature 354:82, 1991; Eichler and Houghten, Biochemistry 32:11035, 1993).In these first two categories, the building blocks are typically L-aminoacids, D-amino acids, unnatural amino acids, or some mixture orcombination thereof. A third category uses molecular biology approachesto prepare peptides or proteins on the surface of filamentous phageparticles or plasmids (Scott and Craig, Curr. Opinion Biotech. 5:40,1994). Soluble, nonsupport-bound peptide libraries appear to be suitablefor a number of applications, including use as tags. The availablerepertoire of chemical diversities in peptide libraries can be expandedby steps such as permethylation (Ostresh et al., Proc. Natl. Acad. Sci.,USA 91:11138, 1994).

Numerous variants of peptide combinatorial libraries are possible inwhich the peptide backbone is modified, and/or the amide bonds have beenreplaced by mimetic groups. Amide mimetic groups which may be usedinclude ureas, urethanes, and carbonylmethylene groups. Restructuringthe backbone such that sidechains emanate from the amide nitrogens ofeach amino acid, rather than the alpha-carbons, gives libraries ofcompounds known as peptoids (Simon et al., Proc. Natl. Acad. Sci., USA89:9367, 1992).

Another common type of oligomeric combinatorial library is theoligonucleotide combinatorial library, where the building blocks aresome form of naturally occurring or unnatural nucleotide orpolysaccharide derivatives, including where various organic andinorganic groups may substitute for the phosphate linkage, and nitrogenor sulfur may substitute for oxygen in an ether linkage (Schneider etal., Biochem. 34:9599, 1995; Freier et al., J. Med. Chem. 38:344, 1995;Frank, J. Biotechnology 41:259, 1995; Schneider et al., Published PCT WO942052; Ecker et al., Nucleic Acids Res. 21:1853, 1993).

More recently, the combinatorial production of collections ofnon-oligomeric, small molecule compounds has been described (DeWitt etal., Proc. Natl. Acad. Sci., USA 90:690, 1993; Bunin et al., Proc. Natl.Acad. Sci., USA 91:4708, 1994). Structures suitable for elaboration intosmall-molecule libraries encompass a wide variety of organic molecules,for example heterocyclics, aromatics, alicyclics, aliphatics, steroids,antibiotics, enzyme inhibitors, ligands, hormones, drugs, alkaloids,opioids, terpenes, porphyrins, toxins, catalysts, as well ascombinations thereof.

g. Specific Methods for Combinatorial Synthesis of Tags

Two methods for the preparation and use of a diverse set ofamine-containing MS tags are outlined below. In both methods, solidphase synthesis is employed to enable simultaneous parallel synthesis ofa large number of tagged linkers, using the techniques of combinatorialchemistry. In the first method, the eventual cleavage of the tag fromthe oligonucleotide results in liberation of a carboxyl amide. In thesecond method, cleavage of the tag produces a carboxylic acid. Thechemical components and linking elements used in these methods areabbreviated as follows:

R = resin FMOC = fluorenylmethoxycarbonyl protecting group All = allylprotecting group CO₂H = carboxylic acid group CONH₂ = carboxylic amidegroup NH₂ = amino group OH = hydroxyl group CONH = amide linkage COO =ester linkage NH₂—Rink—CO₂H = 4-[(α-amino)-2,4-dimethoxybenzyl]-phenoxybutyric acid (Rink linker) OH—1MeO—CO₂H =(4-hydroxymethyl)phenoxybutyric acid OH—2MeO—CO₂H =(4-hydroxymethyl-3-methoxy)phenoxyacetic acid NH₂—A—COOH = amino acidwith aliphatic or aromatic amine functionality in side chain X1 . . .Xn—COOH = set of n diverse carboxylic acids with unique molecularweights oligo1 . . . oligo(n) = set of n oligonucleotides HBTU =O-benzotriazol-1-yl-N,N,N′,N′- tetramethyluronium hexafluorophosphate

The sequence of steps in Method 1 is as follows:

OH-2MeO-CONH-R ↓ FMOC-NH-Rink-CO₂H; couple (e.g, HBTU)FMOC-NH-Rink-COO-2MeO-CONH-R ↓ piperidine (remove FMOC)NH₂-Rink-COO-2MeO-CONH-R ↓ FMOC-NH-A-COOH; couple (e.g., HBTU)FMOC-NH-A-CONH-Rink-COO-2MeO-CONH-R ↓ piperidine (remove FMOC)NH₂-A-CONH-Rink-COO-2MeO-CONH-R ↓ divide into n aliquots ↓↓↓↓↓ couple ton different acids X1 . . . Xn-COOH X1 . . .Xn-CONH-A-CONH-Rink-COO-2MeO-CONH-R ↓↓↓↓↓ Cleave tagged linkers fromresin with 1% TFA X1 . . . Xn-CONH-A-CONH-Rink-CO₂H ↓↓↓↓↓ couple to noligos (oligo1 . . . oligo(n))  (e.g., via Pfp esters) X1 . . .Xn-CONH-A-CONH-Rink-CONH-oligo1 . . . oligo(n) ↓ pool tagged oligos ↓perform sequencing reaction ↓ separate different length fragments from sequencing reaction (e.g., via HPLC or CE) ↓ cleave tags from linkerswith 25%-100% TFA X1 . . . Xn-CONH-A-CONH ↓ analyze by mass spectrometryThe sequence of steps in Method 2 is as follows: OH-1MeO-CO₂-All ↓FMOC-NH-A-CO₂H; couple (e.g., HBTU) FMOC-NH-A-COO-1MeO-CO₂-All ↓Palladium (remove Allyl) FMOC-NH-A-COO-1MeO-CO₂H ↓ OH-2MeO-CONH-R;couple (e.g, HBTU) FMOC-NH-A-COO-1MeO-COO-2MeO-CONH-R ↓ piperidine(remove FMOC) NH₂-A-COO-1MeO-COO-2MeO-CONH-R ↓ divide into n aliquots↓↓↓↓↓↓ couple to n different acids X1 . . . Xn-CO₂H X1 . . .Xn-CONH-A-COO-1MeO-COO-2MeO-CONH-R ↓↓↓↓↓ cleave tagged linkers fromresin with 1% TFA X1 . . . Xn-CONH-A-COO-1MeO-CO₂H ↓↓↓↓↓ couple to noligos (oligo1 . . . oligo(n))  (e.g., via Pfp esters) X1 . . .Xn-CONH-A-COO-1MeO-CONH-oligo1 . . . oligo(n) ↓ pool tagged oligos ↓perform sequencing reaction ↓ separate different length fragments from sequencing reaction (e.g., via HPLC or CE) ↓ cleave tags from linkerswith 25-100% TFA X1 . . . Xn-CONH-A-CO₂H ↓ analyze by mass spectrometry

2. Linkers

A “linker” component (or L), as used herein, means either a directcovalent bond or an organic chemical group which is used to connect a“tag” (or T) to a “molecule of interest” (or MOI) through covalentchemical bonds. In addition, the direct bond itself, or one or morebonds within the linker component is cleavable under conditions whichallows T to be released (in other words, cleaved) from the remainder ofthe T—L—X compound (including the MOI component). The tag variablecomponent which is present within T should be stable to the cleavageconditions. Preferably, the cleavage can be accomplished rapidly; withina few minutes and preferably within about 15 seconds or less.

In general, a linker is used to connect each of a large set of tags toeach of a similarly large set of MOIs. Typically, a single tag-linkercombination is attached to each MOI (to give various T—L—MOI), but insome cases, more than one tag-linker combination may be attached to eachindividual MOI (to give various (T—L)n—MOI). In another embodiment ofthe present invention, two or more tags are bonded to a single linkerthrough multiple, independent sites on the linker, and this multipletag-linker combination is then bonded to an individual MOI (to givevarious (T)n—L—MOI).

After various manipulations of the set of tagged MOIs, special chemicaland/or physical conditions are used to cleave one or more covalent bondsin the linker, resulting in the liberation of the tags from the MOIs.The cleavable bond(s) may or may not be some of the same bonds that wereformed when the tag, linker, and MOI were connected together. The designof the linker will, in large part, determine the conditions under whichcleavage may be accomplished. Accordingly, linkers may be identified bythe cleavage conditions they are particularly susceptible too. When alinker is photolabile (i.e., prone to cleavage by exposure to actinicradiation), the linker may be given the designation L^(hν). Likewise,the designations L^(acid), L^(base), L^([O]), L^([R]), L^(enz), L^(elc),L^(Δ) and L^(SS) may be used to refer to linkers that are particularlysusceptible to cleavage by acid, base, chemical oxidation, chemicalreduction, the catalytic activity of an enzyme (more simply “enzyme”),electrochemical oxidation or reduction, elevated temperature (“thermal”)and thiol exchange, respectively.

Certain types of linker are labile to a single type of cleavagecondition, whereas others are labile to several types of cleavageconditions. In addition, in linkers which are capable of bondingmultiple tags (to give (T)n—L—MOI type structures), each of thetag-bonding sites may be labile to different cleavage conditions. Forexample, in a linker having two tags bonded to it, one of the tags maybe labile only to base, and the other labile only to photolysis.

A linker which is useful in the present invention possesses severalattributes:

1) The linker possesses a chemical handle (L_(h)) through which it canbe attached to an MOI.

2) The linker possesses a second, separate chemical handle (L_(h))through which the tag is attached to the linker. If multiple tags areattached to a single linker ((T)n—L—MOI type structures), then aseparate handle exists for each tag.

3) The linker is stable toward all manipulations to which it issubjected, with the exception of the conditions which allow cleavagesuch that a T-containing moiety is released from the remainder of thecompound, including the MOI. Thus, the linker is stable duringattachment of the tag to the linker, attachment of the linker to theMOI, and any manipulations of the MOI while the tag and linker (T—L) areattached to it.

4) The linker does not significantly interfere with the manipulationsperformed on the MOI while the T—L is attached to it. For instance, ifthe T—L is attached to an oligonucleotide, the T—L must notsignificantly interfere with any hybridization or enzymatic reactions(e.g., PCR) performed on the oligonucleotide. Similarly, if the T—L isattached to an antibody, it must not significantly interfere withantigen recognition by the antibody.

5) Cleavage of the tag from the remainder of the compound occurs in ahighly controlled manner, using physical or chemical processes that donot adversely affect the detectability of the tag.

For any given linker, it is preferred that the linker be attachable to awide variety of MOIs, and that a wide variety of tags be attachable tothe linker. Such flexibility is advantageous because it allows a libraryof T—L conjugates, once prepared, to be used with several different setsof MOIs.

As explained above, a preferred linker has the formula

 L_(h)—L¹—L²—L³—L_(h)

wherein each L_(h) is a reactive handle that can be used to link thelinker to a tag reactant and a molecule of interest reactant. L² is anessential part of the linker, because L² imparts lability to the linker.L¹ and L³ are optional groups which effectively serve to separate L²from the handles L_(h).

L¹ (which, by definition, is nearer to T than is L³), serves to separateT from the required labile moiety L². This separation may be useful whenthe cleavage reaction generates particularly reactive species (e.g.,free radicals) which may cause random changes in the structure of theT-containing moiety. As the cleavage site is further separated from theT-containing moiety, there is a reduced likelihood that reactive speciesformed at the cleavage site will disrupt the structure of theT-containing moiety. Also, as the atoms in L1 will typically be presentin the T-containing moiety, these L¹ atoms may impart a desirablequality to the T-containing moiety. For example, where the T-containingmoiety is a T^(ms)-containing moiety, and a hindered amine is desirablypresent as part of the structure of the T^(ms)-contaiing moiety (toserve, e.g., as a MSSE), the hindered amine may be present in L¹ labilemoiety.

In other instances, L¹ and/or L³ may be present in a linker componentmerely because the commercial supplier of a linker chooses to sell thelinker in a form having such a L¹ and/or L³ group. In such an instance,there is no harm in using linkers having L¹ and/or L³ groups, (so longas these group do not inhibit the cleavage reaction) even though theymay not contribute any particular performance advantage to the compoundsthat incorporate them. Thus, the present invention allows for L¹ and/orL³ groups to be present in the linker component.

L¹ and/or L³ groups may be a direct bond (in which case the group iseffectively not present), a hydrocarbylene group (e.g., alkylene,arylene, cycloalkylene, etc.), —O-hydrocarbylene (e.g., —O—CH₂—,O—CH₂CH(CH₃)—, etc.) or hydrocarbylene-(O-hydrocarbylene)_(w)— wherein wis an integer ranging from 1 to about 10 (e.g., —CH₂—O—Ar—,—CH₂—(O—CH₂CH₂)₄—, etc.).

With the advent of solid phase synthesis, a great body of literature hasdeveloped regarding linkers that are labile to specific reactionconditions. In typical solid phase synthesis, a solid support is bondedthrough a labile linker to a reactive site, and a molecule to besynthesized is generated at the reactive site. When the molecule hasbeen completely synthesized, the solid support-linker-molecule constructis subjected to cleavage conditions which releases the molecule from thesolid support. The labile linkers which have been developed for use inthis context (or which may be used in this context) may also be readilyused as the linker reactant in the present invention.

Lloyd-Williams, P., et al., “Convergent Solid-Phase Peptide Synthesis”,Tetrahedron Report No. 347, 49(48):11065-11133 (1993) provides anextensive discussion of linkers which are labile to actinic radiation(i.e., photolysis), as well as acid, base and other cleavage conditions.Additional sources of information about labile linkers are well known inthe art.

As described above, different linker designs will confer cleavability(“lability”) under different specific physical or chemical conditions.Examples of conditions which serve to cleave various designs of linkerinclude acid, base, oxidation, reduction, fluoride, thiol exchange,photolysis, and enzymatic conditions.

Examples of cleavable linkers that satisfy the general criteria forlinkers listed above will be well known to those in the art and includethose found in the catalog available from Pierce (Rockford, Ill.).Examples include:

ethylene glycobis(succinimidylsuccinate) (EGS), an amine reactivecross-linking reagent which is cleavable by hydroxylamine (1 M at 37° C.for 3-6 hours);

disuccinimidyl tartarate (DST) and sulfo-DST, which are amine reactivecross-linking reagents, cleavable by 0.015 M sodium periodate;

bis[2-(succinimidyloxycarbonyloxy)ethyl]sulfone (BSOCOES) andsulfo-BSOCOES, which are amine reactive cross-linking reagents,cleavable by base (pH 11.6);

1,4-di-[3′-(2′-pyridyldithio(propionamido))butane (DPDPB), apyridyldithiol crosslinker which is cleavable by thiol exchange orreduction;

N-[4-(p-azidosalicylamido)-butyl]-3′-(2′-pyridydithio)propionamide(APDP), a pyridyldithiol crosslinker which is cleavable by thiolexchange or reduction;

bis-[beta-4-(azidosalicylamido)ethyl]-disulfide, a photoreactivecrosslinker which is cleavable by thiol exchange or reduction;

N-succinimidyl-(4-azidophenyl)-1,3′dithiopropionate (SADP), aphotoreactive crosslinker which is cleavable by thiol exchange orreduction;

sulfosuccinimidyl-2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3′-dithiopropionate(SAED), a photoreactive crosslinker which is cleavable by thiol exchangeor reduction;

sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′dithiopropionate(SAND), a photoreactive crosslinker which is cleavable by thiol exchangeor reduction.

Other examples of cleavable linkers and the cleavage conditions that canbe used to release tags are as follows. A silyl linking group can becleaved by fluoride or under acidic conditions. A 3-, 4-, 5-, or6-substituted-2-nitrobenzyloxy or 2-, 3-, 5-, or6-substituted-4-nitrobenzyloxy linking group can be cleaved by a photonsource (photolysis). A 3-, 4-, 5-, or 6-substituted-2-alkoxyphenoxy or2-, 3-, 5-, or 6-substituted-4-alkoxyphenoxy linking group can becleaved by Ce(NH₄)₂(NO₃)₆ (oxidation). A NCO₂ (urethane) linker can becleaved by hydroxide (base), acid, or LiAlH₄ (reduction). A 3-pentenyl,2-butenyl, or 1-butenyl linking group can be cleaved by O₃, O_(S)O₄/IO₄⁻, or KMnO₄ (oxidation). A 2-[3-, 4-, or 5-substituted-furyl]oxy linkinggroup can be cleaved by O₂, Br₂, MeOH, or acid.

Conditions for the cleavage of other labile linking groups include:t-alkyloxy linking groups can be cleaved by acid; methyl(dialkyl)methoxyor 4-substituted-2-alkyl-1,3-dioxlane-2-yl linking groups can be cleavedby H₃O⁺; 2-silylethoxy linking groups can be cleaved by fluoride oracid; 2-(X)-ethoxy (where X=keto, ester amide, cyano, NO₂, sulfide,sulfoxide, sulfone) linking groups can be cleaved under alkalineconditions; 2-, 3-, 4-, 5-, or 6-substituted-benzyloxy linking groupscan be cleaved by acid or under reductive conditions; 2-butenyloxylinking groups can be cleaved by (Ph₃P)₃RhCl(H), 3-, 4-, 5-, or6-substituted-2-bromophenoxy linking groups can be cleaved by Li, Mg, orBuLi; methylthiomethoxy linking groups can be cleaved by Hg²⁺;2-(X)-ethyloxy (where X=a halogen) linking groups can be cleaved by Znor Mg; 2-hydroxyethyloxy linking groups can be cleaved by oxidation(e.g., with Pb(OAc)₄).

Preferred linkers are those that are cleaved by acid or photolysis.Several of the acid-labile linkers that have been developed for solidphase peptide synthesis are useful for linking tags to MOls. Some ofthese linkers are described in a recent review by Lloyd-Williams et al.(Tetrahedron 49:11065-11133, 1993). One useful type of linker is basedupon p-alkoxybenzyl alcohols, of which two, 4-hydroxymethylphenoxyaceticacid and 4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid, arecommercially available from Advanced ChemTech (Louisville, Ky.). Bothlinkers can be attached to a tag via an ester linkage to thebenzylalcohol, and to an amine-containing MOI via an amide linkage tothe carboxylic acid. Tags linked by these molecules are released fromthe MOI with varying concentrations of trifluoroacetic acid. Thecleavage of these linkers results in the liberation of a carboxylic acidon the tag. Acid cleavage of tags attached through related linkers, suchas 2,4-dimethoxy-4′-(carboxymethyloxy)-benzhydrylamine (available fromAdvanced ChemTech in FMOC-protected form), results in liberation of acarboxylic amide on the released tag.

The photolabile linkers useful for this application have also been forthe most part developed for solid phase peptide synthesis (seeLloyd-Williams review).

These linkers are usually based on 2-nitrobenzylesters or2-nitrobenzylamides. Two examples of photolabile linkers that haverecently been reported in the literature are4-(4-(1-Fmoc-amino)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid (Holmesand Jones, J. Org. Chem. 60:2318-2319, 1995) and3-(Fmoc-amino)-3-(2-nitrophenyl)propionic acid (Brown et al., MolecularDiversity 1:4-12, 1995). Both linkers can be attached via the carboxylicacid to an amine on the MOI. The attachment of the tag to the linker ismade by forming an amide between a carboxylic acid on the tag and theamine on the linker. Cleavage of photolabile linkers is usuallyperformed with UV light of 350 nm wavelength at intensities and timesknown to those in the art. Cleavage of the linkers results in liberationof a primary amide on the tag. Examples of photocleavable linkersinclude nitrophenyl glycine esters, exo- and endo-2-benzonorbomeylchlorides and methane sulfonates, and 3-amino-3(2-nitrophenyl) propionicacid. Examples of enzymatic cleavage include esterases which will cleaveester bonds, nucleases which will cleave phosphodiester bonds, proteaseswhich cleave peptide bonds, etc.

A preferred linker component has an ortho-nitrobenzyl structure as shownbelow:

wherein one carbon atom at positions a, b, c, d or e is substituted with—L³—X, and L¹ (which is preferably a direct bond) is present to the leftof N(R¹) in the above structure. Such a linker component is susceptibleto selective photo-induced cleavage of the bond between the carbonlabeled “a” and N(R¹). The identity of R¹ is not typically critical tothe cleavage reaction, however R¹ is preferably selected from hydrogenand hydrocarbyl. The present invention provides that in the abovestructure, —N(R¹)— could be replaced with —O—. Also in the abovestructure, one or more of positions b, c, d or e may optionally besubstituted with alkyl, alkoxy, fluoride, chloride, hydroxyl,carboxylate or amide, where these substituents are independentlyselected at each occurrence.

A further preferred linker component with a chemical handle L_(h) hasthe following structure:

wherein one or more of positions b, c, d or e is substituted withhydrogen, alkyl, alkoxy, fluoride, chloride, hydroxyl, carboxylate oramide, R¹ is hydrogen or hydrocarbyl, and R² is —OH or a group thateither protects or activates a carboxylic acid for coupling with anothermoiety. Fluorocarbon and hydrofluorocarbon groups are preferred groupsthat activate a carboxylic acid toward coupling with another moiety.

3. Molecule of Interest (MOI)

Examples of MOIs include nucleic acids or nucleic acid analogues (e.g.,PNA), fragments of nucleic acids (i.e., nucleic acid fragments),synthetic nucleic acids or fragments, oligonucleotides (e.g., DNA orRNA), proteins, peptides, antibodies or antibody fragments, receptors,receptor ligands, members of a ligand pair, cytokines, hormones,oligosaccharides, synthetic organic molecules, drugs, and combinationsthereof.

Preferred MOIs include nucleic acid fragments. Preferred nucleic acidfragments are primer sequences that are complementary to sequencespresent in vectors, where the vectors are used for base sequencing.Preferably a nucleic acid fragment is attached directly or indirectly toa tag at other than the 3′ end of the fragment; and most preferably atthe 5′ end of the fragment. Nucleic acid fragments may be purchased orprepared based upon genetic databases (e.g., Dib et al., Nature380:152-154, 1996 and CEPH Genotype Database, http://www.cephb.fr) andcommercial vendors (e.g., Promega, Madison, Wis.).

As used herein, MOI includes derivatives of an MOI that containfumctionality useful in joining the MOI to a T—L—L_(h) compound. Forexample, a nucleic acid fragment that has a phosphodiester at the 5′end, where the phosphodiester is also bonded to an alkyleneamine, is anMOI. Such an MOI is described in, e.g., U.S. Pat. No. 4,762,779 which isincorporated herein by reference. A nucleic acid fragment with aninternal modification is also an MOI. An exemplary internal modificationof a nucleic acid fragment is where the base (e.g., adenine, guanine,cytosine, thymidine, uracil) has been modified to add a reactivefunctional group. Such internally modified nucleic acid fragments arecommercially available from, e.g., Glen Research, Herndon, Va. Anotherexemplary internal modification of a nucleic acid fragment is where anabasic phosphoramidate is used to synthesize a modified phosphodiesterwhich is interposed between a sugar and phosphate group of a nucleicacid fragment. The abasic phosphoramidate contains a reactive groupwhich allows a nucleic acid fragment that contains thisphosphoramidate-derived moiety to be joined to another moiety, e.g., aT—L—L_(h) compound. Such abasic phosphoramidates are commerciallyavailable from, e.g., Clonetech Laboratories, Inc., Palo Alto, Calif.

4. Chemical Handles (L_(h))

A chemical handle is a stable yet reactive atomic arrangement present aspart of a first molecule, where the handle can undergo chemical reactionwith a complementary chemical handle present as part of a secondmolecule, so as to form a covalent bond between the two molecules. Forexample, the chemical handle may be a hydroxyl group, and thecomplementary chemical handle may be a carboxylic acid group (or anactivated derivative thereof, e.g., a hydrofluroaryl ester), whereuponreaction between these two handles forms a covalent bond (specifically,an ester group) that joins the two molecules together.

Chemical handles may be used in a large number of covalent bond-formingreactions that are suitable for attaching tags to linkers, and linkersto MOIs. Such reactions include alkylation (e.g., to form ethers,thioethers), acylation (e.g., to form esters, amides, carbamates, ureas,thioureas), phosphorylation (e.g., to form phosphates, phosphonates,phosphoramides, phosphonamides), sulfonylation (e.g., to formsulfonates, sulfonamides), condensation (e.g., to form imines, oximes,hydrazones), silylation, disulfide formation, and generation of reactiveintermediates, such as nitrenes or carbenes, by photolysis. In general,handles and bond-forming reactions which are suitable for attaching tagsto linkers are also suitable for attaching linkers to MOIs, andvice-versa. In some cases, the MOI may undergo prior modification orderivitization to provide the handle needed for attaching the linker.

One type of bond especially useful for attaching linkers to MOIs is thedisulfide bond. Its formation requires the presence of a thiol group(“handle”) on the linker, and another thiol group on the MOI. Mildoxidizing conditions then suffice to bond the two thiols together as adisulfide. Disulfide formation can also be induced by using an excess ofan appropriate disulfide exchange reagent, e.g., pyridyl disulfides.Because disulfide formation is readily reversible, the disulfide mayalso be used as the cleavable bond for liberating the tag, if desired.This is typically accomplished under similarly mild conditions, using anexcess of an appropriate thiol exchange reagent, e.g., dithiothreitol.

Of particular interest for linking tags (or tags with linkers) tooligonucleotides is the formation of amide bonds. Primary aliphaticamine handles can be readily introduced onto synthetic oligonucleotideswith phosphoramidites such as6-monomethoxytritylhexylcyanoethyl-N,N-diisopropyl phosphoramidite(available from Glenn Research, Sterling, Va.). The amines found onnatural nucleotides such as adenosine and guanosine are virtuallyunreactive when compared to the introduced primary amine. Thisdifference in reactivity forms the basis of the ability to selectivelyform amides and related bonding groups (e.g., ureas, thioureas,sulfonamides) with the introduced primary amine, and not the nucleotideamines.

As listed in the Molecular Probes catalog (Eugene, Oreg.), a partialenumeration of amine-reactive functional groups includes activatedcarboxylic esters, isocyanates, isothiocyanates, sulfonyl halides, anddichlorotriazenes. Active esters are excellent reagents for aminemodification since the amide products formed are very stable. Also,these reagents have good reactivity with aliphatic amines and lowreactivity with the nucleotide amines of oligonucleotides. Examples ofactive esters include N-hydroxysuccinimide esters, pentafluorophenylesters, tetrafluorophenyl esters, and p-nitrophenyl esters. Activeesters are useful because they can be made from virtually any moleculethat contains a carboxylic acid. Methods to make active esters arelisted in Bodansky (Principles of Peptide Chemistry (2d ed.), SpringerVerlag, London, 1993).

5. Linker Attachment

Typically, a single type of linker is used to connect a particular setor family of tags to a particular set or family of MOIs. In a preferredembodiment of the invention, a single, uniform procedure may be followedto create all the various T—L—MOI structures. This is especiallyadvantageous when the set of T—L—MOI structures is large, because itallows the set to be prepared using the methods of combinatorialchemistry or other parallel processing technology. In a similar manner,the use of a single type of linker allows a single, uniform procedure tobe employed for cleaving all the various T—L—MOI structures. Again, thisis advantageous for a large set of T—L—MOI structures, because the setmay be processed in a parallel, repetitive, and/or automated manner.

There are, however, other embodiment of the present invention, whereintwo or more types of linker are used to connect different subsets oftags to corresponding subsets of MOIs. In this case, selective cleavageconditions may be used to cleave each of the linkers independently,without cleaving the linkers present on other subsets of MOIs.

A large number of covalent bond-forming reactions are suitable forattaching tags to linkers, and linkers to MOIs. Such reactions includealkylation (e.g., to form ethers, thioethers), acylation (e.g., to formesters, amides, carbamates, ureas, thioureas), phosphorylation (e.g., toform phosphates, phosphonates, phosphoramides, phosphonamides),sulfonylation (e.g., to form sulfonates, sulfonamides), condensation(e.g., to form imines, oximes, hydrazones), silylation, disulfideformation, and generation of reactive intermediates, such as nitrenes orcarbenes, by photolysis. In general, handles and bond-forming reactionswhich are suitable for attaching tags to linkers are also suitable forattaching linkers to MOIs, and vice-versa. In some cases, the MOI mayundergo prior modification or derivitization to provide the handleneeded for attaching the linker.

One type of bond especially useful for attaching linkers to MOIs is thedisulfide bond. Its formation requires the presence of a thiol group(“handle”) on the linker, and another thiol group on the MOI. Mildoxidizing conditions then suffice to bond the two thiols together as adisulfide. Disulfide formation can also be induced by using an excess ofan appropriate disulfide exchange reagent, e.g., pyridyl disulfides.Because disulfide formation is readily reversible, the disulfide mayalso be used as the cleavable bond for liberating the tag, if desired.This is typically accomplished under similarly mild conditions, using anexcess of an appropriate thiol exchange reagent, e.g., dithiothreitol.

Of particular interest for linking tags to oligonucleotides is theformation of amide bonds. Primary aliphatic amine handles can be readilyintroduced onto synthetic oligonucleotides with phosphoramidites such as6-monomethoxytritylhexylcyanoethyl-N,N-diisopropyl phosphoramidite(available from Glenn Research, Sterling, Va.). The amines found onnatural nucleotides such as adenosine and guanosine are virtuallyunreactive when compared to the introduced primary amine. Thisdifference in reactivity forms the basis of the ability to selectivelyform amides and related bonding groups (e.g., ureas, thioureas,sulfonamides) with the introduced primary amine, and not the nucleotideamines.

As listed in the Molecular Probes catalog (Eugene, Oreg.), a partialenumeration of amine-reactive functional groups includes activatedcarboxylic esters, isocyanates, isothiocyanates, sulfonyl halides, anddichlorotriazenes. Active esters are excellent reagents for aminemodification since the amide products formed are very stable. Also,these reagents have good reactivity with aliphatic amines and lowreactivity with the nucleotide amines of oligonucleotides. Examples ofactive esters include N-hydroxysuccinimide esters, pentafluorophenylesters, tetrafluorophenyl esters, and p-nitrophenyl esters. Activeesters are useful because they can be made from virtually any moleculethat contains a carboxylic acid. Methods to make active esters arelisted in Bodansky (Principles of Peptide Chemistry (2d ed.), SpringerVerlag, London, 1993).

Numerous commercial cross-linking reagents exist which can serve aslinkers (e.g., see Pierce Cross-linkers, Pierce Chemical Co., Rockford,Ill.). Among these are homobifunctional amine-reactive cross-linkingreagents which are exemplified by homobiftnctional imidoesters andN-hydroxysuccinimidyl (NHS) esters. There also exist heterobifunctionalcross-linking reagents possess two or more different reactive groupsthat allows for sequential reactions. Imidoesters react rapidly withamines at alkaline pH. NHS-esters give stable products when reacted withprimary or secondary amines. Maleimides, alkyl and aryl halides,alpha-haloacyls and pyridyl disulfides are thiol reactive. Maleimidesare specific for thiol (sulfhydryl) groups in the pH range of 6.5 to7.5, and at alkaline pH can become amine reactive. The thioether linkageis stable under physiological conditions. Alpha-haloacetyl cross-linkingreagents contain the iodoacetyl group and are reactive towardssulfhydryls. Imidazoles can react with the iodoacetyl moiety, but thereaction is very slow. Pyridyl disulfides react with thiol groups toform a disulfide bond. Carbodiimides couple carboxyls to primary aminesof hydrazides which give rises to the formation of an acyl-hydrazinebond. The arylazides are photoaffinity reagents which are chemicallyinert until exposed to UV or visible light. When such compounds arephotolyzed at 250-460 nm, a reactive aryl nitrene is formed. Thereactive aryl nitrene is relatively non-specific. Glyoxals are reactivetowards guanidinyl portion of arginine.

In one typical embodiment of the present invention, a tag is firstbonded to a linker, then the combination of tag and linker is bonded toa MOI, to create the structure T—L—MOI. Alternatively, the samestructure is formed by first bonding a linker to a MOI, and then bondingthe combination of linker and MOI to a tag. An example is where the MOIis a DNA primer or oligonucleotide. In that case, the tag is typicallyfirst bonded to a linker, then the T—L is bonded to a DNA primer oroligonucleotide, which is then used, for example, in a sequencingreaction.

One useful form in which a tag could be reversibly attached to an MOI(e.g., an oligonucleotide or DNA sequencing primer) is through achemically labile linker. One preferred design for the linker allows thelinker to be cleaved when exposed to a volatile organic acid, forexample, trifluoroacetic acid (TFA). TFA in particular is compatiblewith most methods of MS ionization, including electrospray.

As described in detail below, the invention provides a method fordetermining the sequence of a nucleic acid molecule. A composition whichmay be formed by the inventive method comprises a plurality of compoundsof the formula:

T^(ms)—L—MOI

wherein T^(ms) is an organic group detectable by mass spectrometry.T^(ms) contains carbon, at least one of hydrogen and fluoride, and maycontain optional atoms including oxygen, nitrogen, sulfur, phosphorusand iodine. In the formula, L is an organic group which allows aT^(ms)-containing moiety to be cleaved from the remainder of thecompound upon exposure of the compound to cleavage condition. Thecleaved T^(ms)-containing moiety includes a functional group whichsupports a single ionized charge state when each of the plurality ofcompounds is subjected to mass spectrometry. The functional group may bea tertiary amine, quaternary amine or an organic acid. In the formula,MOI is a nucleic acid fragment which is conjugated to L via the 5′ endof the MOI. The term “conjugated” means that there may be chemicalgroups intermediate L and the MOI, e.g., a phosphodiester group and/oran alkylene group. The nucleic acid fragment may have a sequencecomplementary to a portion of a vector, wherein the fragment is capableof priming nucleotide synthesis.

In the composition, no two compounds have either the same T^(ms) or thesame MOI. In other words, the composition includes a plurality ofcompounds, wherein each compound has both a unique T^(ms) and a uniquenucleic acid fragment (unique in that it has a unique base sequence). Inaddition, the composition may be described as having a plurality ofcompounds wherein each compound is defined as having a unique T^(ms),where the T^(ms) is unique in that no other compound has a T^(ms) thatprovides the same signal by mass spectrometry. The composition thereforecontains a plurality of compounds, each having a T^(ms) with a uniquemass. The composition may also be described as having a plurality ofcompounds wherein each compound is defined as having a unique nucleicacid sequence. These nucleic acid sequences are intentionally unique sothat each compound will serve as a primer for only one vector, when thecomposition is combined with vectors for nucleic acid sequencing. Theset of compounds having unique T^(ms) groups is the same set ofcompounds which has unique nucleic acid sequences.

Preferably, the T^(ms) groups are unique in that there is at least a 2amu, more preferably at least a 3 amu, and still more preferably atleast a 4 amu mass separation between the T^(ms) groups of any twodifferent compounds. In the composition, there are at least 2 differentcompounds, preferably there are more than 2 different compounds, andmore preferably there are more than 4 different compounds. Thecomposition may contain 100 or more different compounds, each compoundhaving a unique T^(ms) and a unique nucleic acid sequence.

Another composition that is useful in, e.g., determining the sequence ofa nucleic acid molecule, includes water and a compound of the formulaT^(ms)—L—MOI, wherein T^(ms) is an organic group detectable by massspectrometry. T^(ms) contains carbon, at least one of hydrogen andfluoride, and may contain optional atoms including oxygen, nitrogen,sulfur, phosphorus and iodine. In the formula, L is an organic groupwhich allows a T^(ms)-containing moiety to be cleaved from the remainderof the compound upon exposure of the compound to cleavage condition. Thecleaved T^(ms)-containing moiety includes a functional group whichsupports a single ionized charge state when each of the plurality ofcompounds is subjected to mass spectrometry. The functional group may bea tertiary amine, quaternary amine or an organic acid. In the formula,MOI is a nucleic acid fragment attached at its 5′ end.

In addition to water, this composition may contain a buffer, in order tomaintain the pH of the aqueous composition within the range of about 5to about 9. Furthermore, the composition may contain an enzyme, salts(such as MgCl₂ and NaCl) and one of dATP, dGTP, dCTP, and dTTP. Apreferred composition contains water, T^(ms)—L—MOI and one (and onlyone) of ddATP, ddGTP, ddCTP, and ddTTP. Such a composition is suitablefor use in the dideoxy sequencing method.

The invention also provides a composition which contains a plurality ofsets of compounds, wherein each set of compounds has the formula:

 T^(ms)—L—MOI

wherein T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine. Lis an organic group which allows a T^(ms)-containing moiety to becleaved from the remainder of the compound, wherein theT^(ms)-containing moiety comprises a functional group which supports asingle ionized charge state when the compound is subjected to massspectrometry and is selected from tertiary amine, quaternary amine andorganic acid. The MOI is a nucleic acid fragment wherein L is conjugatedto MOI at the MOI's 5′ end.

Within a set, all members have the same T^(ms) group, and the MOIfragments have variable lengths that terminate with the samedideoxynucleotide selected from ddAMP, ddGMP, ddCMP and ddTMP; andbetween sets, the T^(ms) groups differ by at least 2 amu, preferably byat least 3 amu. The plurality of sets is preferably at least 5 and maynumber 100 or more.

In a preferred composition comprising a first plurality of sets asdescribed above, there is additionally present a second plurality ofsets of compounds having the formula

T^(ms)—L—MOI

wherein T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine. Lis an organic group which allows a T^(ms)-containing moiety to becleaved from the remainder of the compound, wherein theT^(ms)-containing moiety comprises a functional group which supports asingle ionized charge state when the compound is subjected to massspectrometry and is selected from tertiary amine, quaternary amine andorganic acid. MOI is a nucleic acid fragment wherein L is conjugated toMOI at the MOI's 5′ end. All members within the second plurality have anMOI sequence which terminates with the same dideoxynucleotide selectedfrom ddAMP, ddGMP, ddCMP and ddTMP; with the proviso that thedideoxynucleotide present in the compounds of the first plurality is notthe same dideoxynucleotide present in the compounds of the secondplurality.

The invention also provides a kit for DNA sequencing analysis. The kitcomprises a plurality of container sets, where each container setincludes at least five containers. The first container contains avector. The second, third, fourth and fifth containers contain compoundsof the formula:

T^(ms)—L—MOI

wherein T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine. Lis an organic group which allows a T^(ms)-containing moiety to becleaved from the remainder of the compound, wherein theT^(ms)-containing moiety comprises a functional group which supports asingle ionized charge state when the compound is subjected to massspectrometry and is selected from tertiary amine, quaternary amine andorganic acid. MOI is a nucleic acid fragment wherein L is conjugated toMOI at the MOI's 5′ end. The MOI for the second, third, fourth and fifthcontainers is identical and complementary to a portion of the vectorwithin the set of containers, and the T^(ms) group within each containeris different from the other T^(ms) groups in the kit.

Preferably, within the kit, the plurality is at least 3, i.e., there areat least three sets of containers. More preferably, there are at least 5sets of containers.

As noted above, the present invention provides compositions and methodsfor determining the sequence of nucleic acid molecules. Briefly, suchmethods generally comprise the steps of (a) generating tagged nucleicacid fragments which are complementary to a selected nucleic acidmolecule (e.g., tagged fragments) from a first terminus to a secondterminus of a nucleic acid molecule), wherein a tag is correlative witha particular or selected nucleotide, and may be detected by any of avariety of methods, (b) separating the tagged fragments by sequentiallength, (c) cleaving a tag from a tagged fragment, and (d) detecting thetags, and thereby determining the sequence of the nucleic acid molecule.Each of the aspects will be discussed in more detail below.

B. Sequencing Methods and Strategies

As noted above, the present invention provides methods for determiningthe sequence of a nucleic acid molecule. Briefly, tagged nucleic acidfragments are prepared. The nucleic acid fragments are complementary toa selected target nucleic acid molecule. In a preferred embodiment, thenucleic acid fragments are produced from a first terminus to a secondterminus of a nucleic acid molecule, and more preferably from a 5′terminus to a 3′ terminus. In other preferred embodiments, the taggedfragments are generated from 5′-tagged oligonucleotide primers or taggeddideoxynucleotide terminators. A tag of a tagged nucleic acid fragmentis correlative with a particular nucleotide and is detectable byspectrometry (including fluorescence, but preferably other thanfluorescence), or by potentiometry. In a preferred embodiment, at leastfive tagged nucleic acid fragments are generated and each tag is uniquefor a nucleic acid fragment. More specifically, the number of taggedfragments will generally range from about 5 to 2,000. The tagged nucleicacid fragments may be generated from a variety of compounds, includingthose set forth above. It will be evident to one in the art that themethods of the present invention are not limited to use only of therepresentative compounds and compositions described herein.

Following generation of tagged nucleic acid fragments, the taggedfragments are separated by sequential length. Such separation may beperformed by a variety of techniques. In a preferred embodiment,separation is by liquid chromatography (LC) and particularly preferredis HPLC. Next, the tag is cleaved from the tagged fragment. Theparticular method for breaking a bond to release the tag is selectedbased upon the particular type of susceptibility of the bond tocleavage. For example, a light-sensitive bond (i.e., one that breaks bylight) will be exposed to light. The released tag is detected byspectrometry or potentiometry. Preferred detection means are massspectrometry, infrared spectrometry, ultraviolet spectrometry andpotentiostatic amperometry (e.g., with an amperometric detector orcoulemetric detector).

It will be appreciated by one in the art that one or more of the stepsmay be automated, e.g, by use of an instrument. In addition, theseparation, cleavage and detection steps may be performed in acontinuous manner (e.g., continuous flow/continuous fluid path of taggedfragments through separation to cleavage to tag detection). For example,the various steps may be incorporated into a system, such that the stepsare performed in a continuous manner. Such a system is typically in aninstrument or combination of instruments format. For example, taggednucleic acid fragments that are separated (e.g., by HPLC) may flow intoa device for cleavage (e.g., a photo-reactor) and then into a tagdetector (e.g., a mass spectrometer or coulometric or amperometricdetector). Preferably, the device for cleavage is tunable so that anoptimum wavelength for the cleavage reaction can be selected.

It will be apparent to one in the art that the methods of the presentinvention for nucleic acid sequencing may be performed for a variety ofpurposes. For example, such use of the present methods include primarysequence determination for viral, bacterial, prokaryotic and eukaryotic(e.g., mammalian) nucleic acid molecules; mutation detection;diagnostics; forensics; identity; and polymorphism detection.

1. Sequencing Methods

As noted above, compounds including those of the present invention maybe utilized for a variety of sequencing methods, including bothenzymatic and chemical degradation methods. Briefly, the enzymaticmethod described by Sanger (Proc. Natl. Acad. Sci. (USA) 74:5463, 1977)which utilizes dideoxy-terminators, involves the synthesis of a DNAstrand from a single-stranded template by a DNA polymerase. The Sangermethod of sequencing depends on the fact that that dideoxynucleotides(ddNTPs) are incorporated into the growing strand in the same way anormal deoxynucleotides (albeit at a lower efficiency). However, ddNTPsdiffer from normal deoxynucleotides (dNTPs) in that they lack the 3′-OHgroup necessary for chain elongation. When a ddNTP is incorporated intothe DNA chain, the absence the 3′-hydroxy group prevents the formationof a new phosphodiester bond and the DNA fragment is terminated with theddNTP complementary to the base in the template DNA. The Maxam andGilbert method (Maxam and Gilbert, Proc. Natl. Acad. Sci. (USA) 74:560,1977) employs a chemical degradation method of the original DNA (in bothcases the DNA must be clonal). Both methods produce populations offragments that begin from a particular point and terminate in every basethat is found in the DNA fragment that is to be sequenced. Thetermination of each fragment is dependent on the location of aparticular base within the original DNA fragment. The DNA fragments areseparated by polyacrylamide gel electrophoresis and the order of the DNAbases (A,C,T,G) is read from a autoradiograph of the gel.

2. Exonuclease DNA Sequencing

A procedure for determining DNA nucleotide sequences was reported byLabeit et al. (S. Labeit, H. Lehrach & R. S. Goody, DNA 5: 173-7, 1986;A new method of DNA sequencing using deoxynucleosidealpha-thiotriphosphates). In the first step of the method, four DNAs,each separately substituted with a different deoxynucleosidephosphorothioate in place of the corresponding monophosphate, areprepared by template-directed polymerization catalyzed by DNApolymerase. In the second step, these DNAs are subjected to stringentexonuclease III treatment, which produces only fragments terminatingwith a phosphorothioate internucleotide linkage. These can then beseparated by standard gel electrophoresis techniques and the sequencecan be read directly as in presently used sequencing methods. Porter etal. (K. W. Porter, J. Tomasz, F. Huang, A. Sood & B. R. Shaw,Biochemistry 34: 11963-11969, 1995; N7-cyanoborane-2′-deoxyguanosine5′-triphosphate is a good substrate for DNA polymerase) described a newset of boron-substituted nucleotide analogs which are also exonucleaseresistant and good substrates for a number of polymerases: these baseare also suitable for exonuclease DNA sequencing.

3. A Simplified Strategy for Sequencing Large Numbers of Full LengthcDNAs

cDNA sequencing has been suggested as an alternative to generating thecomplete human genomic sequence. Two approaches have been attempted. Thefirst involves generation of expressed sequence tags (ESTs) through asingle DNA sequence pass at one end of each cDNA clone. This method hasgiven insights into the distribution of types of expressed sequences andhas revealed occasional useful homology with genomic fragments, butoverall has added little to our knowledge base since insufficient datafrom each clone is provided. The second approach is to generate completecDNA sequence which can indicate the possible function of the cDNAs.Unfortunately most cDNAs are of a size range of 1-4 kilobases whichhinders the automation of full-length sequence determination. Currentlythe most efficient method for large scale, high throughput sequenceproduction is from sequencing from a vector/primer site, which typicallyyields less than 500 bases of sequence from each flank. The synthesis ofnew oligonucleotide primers of length 15-18 bases for ‘primer walking’can allow closure of each sequence. An alternative strategy for fulllength cDNA sequencing is to generate modified templates that aresuitable for sequencing with a universal primer, but provide overlappingcoverage of the molecules.

Shotgun sequencing methods can be applied to cDNA sequencing studies bypreparing a separate library from each cDNA clone. These methods havenot been used extensively for the analysis of the 1.5-4.0 kilobasefragments, however, as they are very labor intensive during the initialcloning phase. Instead they have generally been applied to projectswhere the target sequence is of the order of 15 to 40 kilobases, such asin lambda or cosmid inserts.

4. Analogy of cDNA with Genomic Sequencing

Despite the typically different size of the individual clones to beanalyzed in cDNA sequencing, there are similarities with therequirements for large scale genomic DNA sequencing. In addition to alow cost per base, and a high throughput, the ideal strategy for fulllength cDNA sequencing will have a high accuracy. The favored currentmethodology for genomic DNA sequencing involves the preparation ofshotgun sequencing libraries from cosmids, followed by random sequencingusing ABI fluorescent DNA sequencing instruments, and closure(finishing) by directed efforts. Overall there is agreement that thefluorescent shotgun approach is superior to current alternatives interms of efficiency and accuracy. The initial shotgun library quality isa critical determinant of the ease and quality of sequence assembly. Thehigh quality of the available shotgun library procedure has prompted astrategy for the production of multiplex shotgun libraries containingmixtures of the smaller cDNA clones. Here the individual clones to besequenced are mixed prior to library construction and then identifiedfollowing random sequencing, at the stage of computer analysis.Junctions between individual clones are labeled during libraryproduction either by PCR or by identification of vector arm sequence.

Clones may be prepared both by microbial methods or by PCR. When usingPCR, three reactions from each clone are used in order to minimize therisk for errors.

One pass sequencing is a new technique designed to speed theidentification of important sequences within a new region of genomicDNA. Briefly, a high quality shotgun library is prepared and then thesequences sampled to obtain 80-95% coverage. For a cosmid this wouldtypically be about 200 samples. Essentially all genes are likely to haveat least one exon detected in this sample using either sequencesimilarity (BLAST) or exon structure (GRAIL2) screening.

“Skimming” has been successfully applied to cosmids and P1s. One passsequencing is potentially the fastest and least expensive way to findgenes in a positional cloning project. The outcome is virtually assured.Most investigators are currently developing cosmid contigs for exontrapping and related techniques. Cosmids are completely suitable forsequence skimming. P1 and other BACs could be considerably cheaper sincethere is savings both in shotgun library construction and minimizationof overlaps.

5. Shotgun Sequencing

Shotgun DNA sequencing starts with random fragmentation of the targetDNA. Random sequencing is then used to generate the majority of thedata. A directed phase then completes gaps, ensuring coverage of eachstrand in both directions. Shotgun sequencing offers the advantage ofhigh accuracy at relatively low cost. The procedure is best suited tothe analysis of relatively large fragments and is the method of choicein large scale genomic DNA sequencing.

There are several factors that are important in making shotgunsequencing accurate and cost effective. A major consideration is thequality of the shotgun library that is generated, since any clones thatdo not have inserts, or have chimeric inserts, will result in subsequentinefficient sequencing. Another consideration is the careful balancingof the random and the directed phases of the sequencing, so that highaccuracy is obtained with a minimal loss of efficiency throughunnecessary sequencing.

6. Sequencing Chemistry: Tagged-Terminator Chemistry

There are two types of fluorescent sequencing chemistries currentlyavailable: dye primer, where the primer is fluorescently labeled, anddye terminator, where the dideoxy terminators are labeled. Each of thesechemistries can be used with either Taq DNA polymerase or sequenaseenzymes. Sequenase enzyme seems to read easily through G-C rich regions,palindromes, simple repeats and other difficult to read sequences.Sequenase is also good for sequencing mixed populations. Sequenasesequencing requires 5 μg of template, one extension and a multi-stepcleanup process. Tagged-primer sequencing requires four separatereactions, one for each of A, C, G and T and then a laborious cleanupprotocol. Taq terminator cycle sequencing chemistry is the most robustsequencing method. With this method any sequencing primer can be used.The amount of template needed is relatively small and the whole reactionprocess from setup to cleanup is reasonably easy, compared to sequenaseand dye primer chemistries. Only 1.5 μg of DNA template and 4 pm ofprimer are needed. To this a ready reaction mix is added. This mixconsists of buffer, enzyme, dNTPs and labeled dideoxynucleotides. Thisreaction can be done in one tube as each of the four dideoxies islabeled with a different fluorescent dye. These labeled terminators arepresent in this mix in excess because they are difficult to incorporateduring extension. With unclean DNA the incorporation of these highmolecular weight dideoxies can be inhibited. The premix includes dITP tominimize band compression. The use of Taq as the DNA polymerase allowsthe reactions to be run at high temperatures to minimize secondarystructure problems as well as non-specific primer binding. The wholecocktail goes through 25 cycles of denaturation, annealing and extensionin a thermal cycler and the completed reaction is spun through aSephadex G50 (Pharmacia, Piscataway, N.J.) column and is ready for gelloading after five minutes in a vacuum dessicator.

7. Designing Primers

When designing primers, the same criteria should be used as fordesigning PCR primers. In particular, primers should preferably be 18 to20 nucleotides long and the 3-prime end base should be a G or a C.Primers should also preferably have a Tm of more than 50° C. Primersshorter than 18 nucleotides will work but are not recommended. Theshorter the primer the greater the probability of it binding at morethan one site on the template DNA, and the lower its Tm. The sequenceshould have 100% match with the template. Any mismatch, especiallytowards the 3-prime end will greatly diminish sequencing ability.However primers with 5-prime tails can be used as long as there is about18 bases at 3-prime that bind. If one is designing a primer from asequence chromatogram, an area with high confidence must be used. As onemoves out past 350 to 400 bases on a standard chromatogram, the peaksget broader and the base calls are not as accurate. As described herein,the primer may possess a 5′ handle through which a linker or linker tagmay be attached.

8. Nucleic Acid Template Preparation

The most important factor in tagged-primer DNA sequencing is the qualityof the template. Briefly, one common misconception is that if a templateworks in manual sequencing, it should work in automated sequencing. Infact, if a reaction works in manual sequencing it may work in automatedsequencing, however, automated sequencing is much more sensitive and apoor quality template may result in little or no data when fluorescentsequencing methods are utilized. High salt concentrations and other cellmaterial not properly extracted during template preparation, includingRNA, may likewise prevent the ability to obtain accurate sequenceinformation. Many mini and maxi prep protocols produce DNA which is goodenough for manual sequencing or PCR, but not for automated(tagged-primer) sequencing. Also the use of phenol is not at allrecommended as phenol can intercalate in the helix structure. The use of100% chloroform is sufficient. There are a number of DNA preparationmethods which are particularly preferred for the tagged primersequencing methods provided herein. In particular, maxi preps whichutilize cesium chloride preparations or Qiagen (Chatsworth, Calif.) maxiprep. columns (being careful not to overload) are preferred. For minipreps, columns such as Promega's Magic Mini prep (Madison, Wis.), may beutilized. When sequencing DNA fragments such as PCR fragments orrestriction cut fragments, it is generally preferred to cut the desiredfragment from a low melt argarose gel and then purify with a productsuch as GeneClean (La Jolla, Calif.). It is very important to make surethat only one band is cut from the gel. For PCR fragments the PCRprimers or internal primers can be used in order to ensure that theappropriate fragment was sequenced. To get optimum performance from thesequence analysis software, fragments should be larger than 200 bases.Double stranded or single stranded DNA can be sequenced by this method.

An additional factor generally taken into account when preparing DNA forsequencing is the choice of host strain. Companies selling equipment andreagents for sequencing, such as ABI (Foster City, Calif.) and Qiagen(Chatsworth, Calif.), typically recommend preferred host strains, andhave previously recommended strains such as DH5 alpha, HB101, XL-1 Blue,JM109, MV1190. Even when the DNA preparations are very clean, there areother inherent factors which can make it difficult to obtain sequence.G-C rich templates are always difficult to sequence through, andsecondary structure can also cause problems. Sequencing through a longrepeats often proves to be difficult. For instance as Taq moves along apoly T stretch, the enzyme often falls off the template and jumps backon again, skipping a T. This results in extension products with X amountof Ts in the poly T stretch and fragments with X-1, X-2 etc. amounts ofTs in the poly T stretch. The net effect is that more than one baseappears in each position making the sequence impossible to read.

9. Use of Molecularly Distinct Cloning Vectors

Sequencing may also be accomplished utilizing universal cloning vector(M13) and complementary sequencing primers. Briefly, for present cloningvectors the same primer sequence is used and only 4 tags are employed(each tag is a different fluorophore which represents a differentterminator (ddNTP)), every amplification process must take place indifferent containers (one DNA sample per container). That is, it isimpossible to mix two or different DNA samples in the same amplificationprocess. With only 4 tags available, only one DNA sample can be run pergel lane. There is no convenient means to deconvolute the sequence ofmore than one DNA sample with only 4 tags. (In this regard, workers inthe field take great care not to mix or contaminate different DNAsamples when using current technologies.)

A substantial advantage is gained when multiples of 4 tags can be runper gel lane or respective separation process. In particular, utilizingtags of the present invention, more than one DNA sample in a singleamplification reaction or container can be processed. When multiples of4 tags are available for use, each tag set can be assigned to aparticular DNA sample that is to be amplified. (A tag set is composed ofa series of 4 different tags each with a unique property. Each tag isassigned to represent a different dideoxy-terminator, ddATP, ddGTP,ddCTP, or ddTTP. To employ this advantage a series of vectors must begenerated in which a unique priming site is inserted. A unique primingsite is simply a stretch of 18 nucleotides which differs from vector tovector. The remaining nucleotide sequence is conserved from vector tovector. A sequencing primer is prepared (synthesized) which correspondsto each unique vector. Each unique primer is derived (or labeled) with aunique tag set.

With these respective molecular biology tools in hand, it is possible inthe present invention to process multiple samples in a single container.First, DNA samples which are to be sequenced are cloned into themultiplicity of vectors. For example, if 100 unique vectors areavailable, 100 ligation reactions, plating steps, and picking of plaquesare performed. Second, one sample from each vector type is pooled makinga pool of 100 unique vectors containing 100 unique DNA fragments orsamples. A given DNA sample is therefore identified and automaticallyassigned a primer set with the associated tag set. The respectiveprimers, buffers, polymerase(s), ddNTPs, dNTPs and co-factors are addedto the reaction container and the amplification process is carried out.The reaction is then subjected to a separation step and the respectivesequence is established from the temporal appearance of tags. Theability to pool multiple DNA samples has substantial advantages. Thereagent cost of a typical PCR reaction is about $2.00 per sample. Withthe method described herein the cost of amplification on a per samplebasis could be reduced at least by a factor of 100. Sample handlingcould be reduced by a factor of at least 100, and materials costs couldbe reduced. The need for large scale amplification robots would beobviated.

10. Sequencing Vectors for Cleavable Mass Spectroscopy Tagging

Using cleavable mass spectroscopy tagging (CMST) of the presentinvention, each individual sequencing reaction can be read independentlyand simultaneously as the separation proceeds. In CMST sequencing, adifferent primer is used for each cloning vector: each reaction has 20different primers when 20 clones are used per pool. Each primercorresponds to one of the vectors, and each primer is tagged with aunique CMST molecule. Four reactions are performed on each pooled DNAsample (one for each base), so every vector has four oligonucleotideprimers, each one identical in sequence but tagged with a different CMStag. The four separate sequencing reactions are pooled and run together.When 20 samples are pooled, 80 tags are used (4 bases per sample times20 samples), and all 80 are detected simultaneously as the gel is run.

The construction of the vectors may be accomplished by cloning a random20-mer on either side of a restriction site. The resulting clones aresequenced and a number chosen for use as vectors. Two oligonucleotidesare prepared for each vector chosen, one homologous to the sequence ateach side of the restriction site, and each orientated so that the3′-end is towards the restriction site. Four tagged preparations of eachprimer are prepared, one for each base in the sequencing reactions andeach one labeled with a unique CMS tag.

11. Advantages of Sequencing by the Use of Reversible Tags

There are substantial advantages when cleavable tags are used insequencing and related technologies. First, an increase in sensitivitywill contribute to longer read lengths, as will the ability to collecttags for a specified period of time prior to measurement. The use ofcleavable tags permits the development of a system that equalizesbandwidth over the entire range of the gel (1-1500 nucleotides (nt), forexample). This will greatly impact the ability to obtain read lengthsgreater than 450 nt.

The use of cleavable multiple tags (MW identifiers) also has theadvantage that multiple DNA samples can be run on a single gel lane orseparation process. For example, it is possible using the methodologiesdisclosed herein to combine at least 96 samples and 4 sequencingreactions (A,G,T,C) on a single lane or fragment sizing process. Ifmultiple vectors are employed which possess unique priming sites, thenat least 384 samples can be combined per gel lane (the differentterminator reactions cannot be amplified together with this scheme).When the ability to employ cleavable tags is combined with the abilityto use multiple vectors, an apparent 10,000-fold increase in DNAsequencing throughput is achieved. Also, in the schemes describedherein, reagent use is decreased, disposables decrease, with a resultantdecrease in operating costs to the consumer.

An additional advantage is gained from the ability to process internalcontrols throughout the entire methodologies described here. For any setof samples, an internal control nucleic acid can be placed in thesample(s). This is not possible with the current configurations. Thisadvantage permits the control of the amplification process, theseparation process, the tag detection system and sequence assembly. Thisis an immense advantage over current systems in which the controls arealways separated from the samples in all steps.

The compositions and methods described herein also have the advantagethat they are modular in nature and can be fitted on any type ofseparation process or method and in addition, can be fitted onto anytype of detection system as improvements are made in either types ofrespective technologies. For example, the methodologies described hereincan be coupled with “bundled” CE arrays or microfabricated devices thatenable separation of DNA fragments.

C. Separation of DNA Fragments

A sample that requires analysis is often a mixture of many components ina complex matrix. For samples containing unknown compounds, thecomponents must be separated from each other so that each individualcomponent can be identified by other analytical methods. The separationproperties of the components in a mixture are constant under constantconditions, and therefore once determined they can be used to identifyand quantify each of the components. Such procedures are typical inchromatographic and electrophoretic analytical separations.

1. High-Performance Liquid Chromatography (HPLC)

High-Performance liquid chromatography (HPLC) is a chromatographicseparations technique to separate compounds that are dissolved insolution. HPLC instruments consist of a reservoir of mobile phase, apump, an injector, a separation columni, and a detector. Compounds areseparated by injecting an aliquot of the sample mixture onto the column.The different components in the mixture pass through the column atdifferent rates due to differences in their partitioning behaviorbetween the mobile liquid phase and the stationary phase.

Recently, IP-RO-HPLC on non-porous PS/DVB particles with chemicallybonded alkyl chains have been shown to be rapid alternatives tocapillary electrophoresis in the analysis of both single anddouble-strand nucleic acids providing similar degrees of resolution(Huber et al, 1993, Anal. Biochem., 212, p351; Huber et al., 1993, Nuc.Acids Res., 21, p1061; Huber et al., 1993, Biotechniques, 16, p898). Incontrast to ion-exchange chromatography, which does not always retaindouble-strand DNA as a function of strand length (Since AT base pairsinteract with the positively charged stationary phase, more stronglythan GC base-pairs), IP-RP-HPLC enables a strictly size-dependentseparation.

A method has been developed using 100 mM triethylammonium acetate asion-pairing reagent, phosphodiester oligonucleotides could besuccessfully separated on alkylated non-porous 2.3 μMpoly(styrene-divinylbenzene) particles by means of high performanceliquid chromatography (Oefner et al., 1994, Anal. Biochem., 223, p39).The technique described allowed the separation of PCR products differingonly 4 to 8 base pairs in length within a size range of 50 to 200nucleotides.

2. Electrophoresis

Electrophoresis is a separations technique that is based on the mobilityof ions (or DNA as is the case described herein) in an electric field.Negatively charged DNA charged migrate towards a positive electrode andpositively-charged ions migrate toward a negative electrode. For safetyreasons one electrode is usually at ground and the other is biasedpositively or negatively. Charged species have different migration ratesdepending on their total charge, size, and shape, and can therefore beseparated.

An electrode apparatus consists of a high-voltage power supply,electrodes, buffer, and a support for the buffer such as apolyacrylamide gel, or a capillary tube. Open capillary tubes are usedfor many types of samples and the other gel supports are usually usedfor biological samples such as protein mixtures or DNA fragments.

3. Capillary Electrophoresis (CE)

Capillary electrophoresis (CE) in its various manifestations (freesolution, isotachophoresis, isoelectric focusing, polyacrylamide gel,micellar electrokinetic “chromatography”) is developing as a method forrapid high resolution separations of very small sample volumes ofcomplex mixtures. In combination with the inherent sensitivity andselectivity of MS, CE-MS is a potential powerful technique forbioanalysis. In the novel application disclosed herein, the interfacingof these two methods will lead to superior DNA sequencing methods thateclipse the current rate methods of sequencing by several orders ofmagnitude.

The correspondence between CE and electrospray ionization (ESI) flowrates and the fact that both are facilitated by (and primarily used for)ionic species in solution provide the basis for an extremely attractivecombination. The combination of both capillary zone electrophoresis(CZE) and capillary isotachophoresis with quadrapole mass spectrometersbased upon ESI have been described (Olivares et al., Anal. Chem.59:1230, 1987; Smith et al., Anal. Chem. 60:436, 1988; Loo et al., Anal.Chem. 179:404, 1989; Edmonds et al., J. Chroma. 474:21, 1989; Loo etal., J. Microcolumn Sep. 1:223, 1989; Lee et al., J. Chromatog. 458:313,1988; Smith et al., J. Chromatog. 480:211, 1989; Grese et al., J. Am.Chem. Soc. 111:2835, 1989). Small peptides are easily amenable to CZEanalysis with good (femtomole) sensitivity.

The most powerful separation method for DNA fragments is polyacrylamidegel electrophoresis (PAGE), generally in a slab gel format. However, themajor limitation of the current technology is the relatively long timerequired to perform the gel electrophoresis of DNA fragments produced inthe sequencing reactions. An increase magnitude (10-fold) can beachieved with the use of capillary electrophoresis which utilizeultrathin gels. In free solution to a first approximation all DNAmigrate with the same mobility as the addition of a base results in thecompensation of mass and charge. In polyacrylamide gels, DNA fragmentssieve and migrate as a function of length and this approach has now beenapplied to CE. Remarkable plate number per meter has now been achievedwith cross-linked polyacrylamide (10⁺⁷ plates per meter, Cohen et al.,Proc. Natl. Acad. Sci., USA 85:9660, 1988). Such CE columns as describedcan be employed for DNA sequencing. The method of CE is in principle 25times faster than slab gel electrophoresis in a standard sequencer. Forexample, about 300 bases can be read per hour. The separation speed islimited in slab gel electrophoresis by the magnitude of the electricfield which can be applied to the gel without excessive heat production.Therefore, the greater speed of CE is achieved through the use of higherfield strengths (300 V/cm in CE versus 10 V/cm in slab gelelectrophoresis). The capillary format reduces the amperage and thuspower and the resultant heat generation.

Smith and others (Smith et al., Nuc. Acids. Res. 18:4417, 1990) havesuggested employing multiple capillaries in parallel to increasethroughput. Likewise, Mathies and Huang (Mathies and Huang, Nature359:167, 1992) have introduced capillary electrophoresis in whichseparations are performed on a parallel array of capillaries anddemonstrated high through-put sequencing (Huang et al., Anal. Chem.64:967, 1992, Huang et al., Anal. Chem. 64:2149, 1992). The majordisadvantage of capillary electrophoresis is the limited amount ofsample that can be loaded onto the capillary. By concentrating a largeamount of sample at the beginning of the capillary, prior to separation,loadability is increased, and detection levels can be lowered severalorders of magnitude. The most popular method of preconcentration in CEis sample stacking. Sample stacking has recently been reviewed (Chienand Burgi, Anal. Chem. 64:489A, 1992). Sample stacking depends of thematrix difference, (pH, ionic strength) between the sample buffer andthe capillary buffer, so that the electric field across the sample zoneis more than in the capillary region. In sample stacking, a large volumeof sample in a low concentration buffer is introduced forpreconcentration at the head of the capillary column. The capillary isfilled with a buffer of the same composition, but at higherconcentration. When the sample ions reach the capillary buffer and thelower electric field, they stack into a concentrated zone. Samplestacking has increased detectabilities 1-3 orders of magnitude.

Another method of preconcentration is to apply isotachophoresis (ITP)prior to the free zone CE separation of analytes. ITP is anelectrophoretic technique which allows microliter volumes of sample tobe loaded on to the capillary, in contrast to the low nL injectionvolumes typically associated with CE. The technique relies on insertingthe sample between two buffers (leading and trailing electrolytes) ofhighel and lower mobility respectively, than the analyte. The techniqueis inherently a concentration technique, where the analytes concentrateinto pure zones migrating with the same speed. The technique iscurrently less popular than the stacking methods described above becauseof the need for several choices of leading and trailing electrolytes,and the ability to separate only cationic or anionic species during aseparation process.

The heart of the DNA sequencing process is the remarkably selectiveelectrophoretic separation of DNA or oligonucleotide fragments. It isremarkable because each fragment is resolved and differs by onlynucleotide. Separations of up to 1000 fragments (1000 bp) have beenobtained. A further advantage of sequencing with cleavable tags is asfollows. There is no requirement to use a slab gel format when DNAfragments are separated by polyacrylamide gel electrophoresis whencleavable tags are employed. Since numerous samples are combined (4 to2000) there is no need to run samples in parallel as is the case withcurrent dye-primer or dye-terminator methods (i.e., ABI373 sequencer).Since there is no reason to run parallel lanes, there is no reason touse a slab gel. Therefore, one can employ a tube gel format for theelectrophoretic separation method. Grossman (Grossman et al., Genet.Anal. Tech. Appl. 9:9, 1992) have shown that considerable advantage isgained when a tube gel format is used in place of a slab gel format.This is due to the greater ability to dissipate Joule heat in a tubeformat compared to a slab gel which results in faster run times (by50%), and much higher resolution of high molecular weight DNA fragments(greater than 1000 nt). Long reads are critical in genomic sequencing.Therefore, the use of cleavable tags in sequencing has the additionaladvantage of allowing the user to employ the most efficient andsensitive DNA separation method which also possesses the highestresolution.

4. Microfabricated Devices

Capillary electrophoresis (CE) is a powerful method for DNA sequencing,forensic analysis, PCR product analysis and restriction fragment sizing.CE is far faster than traditional slab PAGE since with capillary gels afar higher potential field can be applied. However, CE has the drawbackof allowing only one sample to be processed per gel. The method combinesthe faster separations times of CE with the ability to analyze multiplesamples in parallel. The underlying concept behind the use ofmicrofabricated devices is the ability to increase the informationdensity in electrophoresis by miniaturizing the lane dimension to about100 micrometers. The electronics industry routinely usesmicrofabrication to make circuits with features of less than one micronin size. The current density of capillary arrays is limited the outsidediameter of the capillary tube. Microfabrication of channels produces ahigher density of arrays. Microfabrication also permits physicalassemblies not possible with glass fibers and links the channelsdirectly to other devices on a chip. Few devices have been constructedon microchips for separation technologies. A gas chromatograph (Terry etal., IEEE Trans. Electron Device, ED-26:1880, 1979) and a liquidchromatograph (Manz et al., Sens. Actuators B1:249, 1990) have beenfabricated on silicon chips, but these devices have not been widelyused. Several groups have reported separating fluorescent dyes and aminoacids on microfabricated devices (Manz et al., J. Chromatography593:253, 1992, Effenhauser et al., Anal. Chem. 65:2637, 1993). RecentlyWoolley and Mathies (Woolley and Mathies, Proc. Natl. Acad. Sci.91:11348, 1994) have shown that photolithography and chemical etchingcan be used to make large numbers of separation channels on glasssubstrates. The channels are filled with hydroxyethyl cellulose (HEC)separation matrices. It was shown that DNA restriction fragments couldbe separated in as little as two minutes.

D. Cleavage of Tags

As described above. different linker designs will confer cleavability(“lability”) under different specific physical or chemical conditions.Examples of conditions which serve to cleave various designs of linkerinclude acid, base, oxidation, reduction, fluoride, thiol exchange,photolysis, and enzymatic conditions.

Examples of cleavable linkers that satisfy the general criteria forlinkers listed above will be well known to those in the art and includethose found in the catalog available from Pierce (Rockford, Ill.).Examples include:

ethylene glycobis(succinimidylsuccinate) (EGS), an amine reactivecross-linking reagent which is cleavable by hydroxylamine (1 M at 37° C.for 3-6 hours);

disuccinimidyl tartarate (DST) and sulfo-DST, which are amine reactivecross-linking reagents, cleavable by 0.015 M sodium periodate;

bis[2-(succinimidyloxycarbonyloxy)ethyl]sulfone (BSOCOES) andsulfo-BSOCOES, which are amine reactive cross-linking reagents,cleavable by base (pH 11.6);

1,4-di-[3′-(2′-pyridyldithio(propionamido))butane (DPDPB), apyridyldithiol crosslinker which is cleavable by thiol exchange orreduction;

N-[4-(p-azidosalicylamido)-butyl]-3′-(2′-pyridydithio)propionamide(APDP), a pyridyldithiol crosslinker which is cleavable by thiolexchange or reduction;

bis-[beta-4-(azidosalicylamido)ethyl]-disulfide, a photoreactivecrosslinker which is cleavable by thiol exchange or reduction;

N-succinimidyl-(4-azidophenyl)-1,3′dithiopropionate (SADP), aphotoreactive crosslinker which is cleavable by thiol exchange orreduction;

sulfosuccinimidyl-2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3′-dithiopropionate(SAED), a photoreactive crosslinker which is cleavable by thiol exchangeor reduction;

sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′dithiopropionate(SAND), a photoreactive crosslinker which is cleavable by thiol exchangeor reduction.

Other examples of cleavable linkers and the cleavage conditions that canbe used to release tags are as follows. A silyl linking group can becleaved by fluoride or under acidic conditions. A 3-, 4-, 5-, or6-substituted-2-nitrobenzyloxy or 2-, 3-, 5-, or6-substituted-4-nitrobenzyloxy linking group can be cleaved by a photonsource (photolysis). A 3-, 4-, 5-, or 6-substituted-2-alkoxyphenoxy or2-, 3-, 5-, or 6-substituted-4-alkoxyphenoxy linking group can becleaved by Ce(NH₄)₂(NO₃)₆ (oxidation). A NCO₂ (urethane) linker can becleaved by hydroxide (base), acid, or LiAlH₄ (reduction). A 3-pentenyl,2-butenyl, or 1-butenyl linking group can be cleaved by O₃, O_(S)O₄/IO₄⁻, or KMnO₄ (oxidation). A 2-[3-, 4-, or 5-substituted-furyl]oxy linkinggroup can be cleaved by O₂, Br₂, MeOH, or acid.

Conditions for the cleavage of other labile linking groups include:t-alkyloxy linking groups can be cleaved by acid; methyl(dialkyl)methoxyor 4-substituted-2-alkyl-1,3-dioxlane-2-yl linking groups can be cleavedby H₃O⁺; 2-silylethoxy linking groups can be cleaved by fluoride oracid; 2-(X)-ethoxy (where X=keto, ester amide, cyano, NO₂, sulfide,sulfoxide, sulfone) linking groups can be cleaved under alkalineconditions; 2-, 3-, 4-, 5-, or 6-substituted-benzyloxy linking groupscan be cleaved by acid or under reductive conditions; 2-butenyloxylinking groups can be cleaved by (Ph₃P)₃RhCl(H), 3-, 4-, 5-, or6-substituted-2-bromophenoxy linking groups can be cleaved by Li, Mg, orBuLi; methylthiomethoxy linking groups can be cleaved by Hg²⁺;2-(X)-ethyloxy (where X=a halogen) linking groups can be cleaved by Znor Mg; 2-hydroxyethyloxy linking groups can be cleaved by oxidation(e.g., with Pb(OAc)₄).

Preferred linkers are those that are cleaved by acid or photolysis.Several of the acid-labile linkers that have been developed for solidphase peptide synthesis are useful for linking tags to MOIs. Some ofthese linkers are described in a recent review by Lloyd-Williams et al.(Tetrahedron 49:11065-11133, 1993). One useful type of linker is basedupon p-alkoxybenzyl alcohols, of which two, 4-hydroxymethylphenoxyaceticacid and 4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid, arecommercially available from Advanced ChemTech (Louisville, Ky.). Bothlinkers can be attached to a tag via an ester linkage to thebenzylalcohol, and to an amine-containing MOI via an amide linkage tothe carboxylic acid. Tags linked by these molecules are released fromthe MOI with varying concentrations of trifluoroacetic acid. Thecleavage of these linkers results in the liberation of a carboxylic acidon the tag. Acid cleavage of tags attached through related linkers, suchas 2,4-dimethoxy-4′-(carboxymethyloxy)-benzhydrylamine (available fromAdvanced ChemTech in FMOC-protected form), results in liberation of acarboxylic amide on the released tag.

The photolabile linkers useful for this application have also been forthe most part developed for solid phase peptide synthesis (seeLloyd-Williams review). These linkers are usually based on2-nitrobenzylesters or 2-nitrobenzylamides. Two examples of photolabilelinkers that have recently been reported in the literature are4-(4-(1-Fmoc-amino)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid (Holmesand Jones, J. Org. Chem. 60:2318-2319, 1995) and3-(Fmoc-amino)-3-(2-nitrophenyl)propionic acid (Brown et al., MolecularDiversity 1:4-12, 1995). Both linkers can be attached via the carboxylicacid to an amine on the MOI. The attachment of the tag to the linker ismade by forming an amide between a carboxylic acid on the tag and theamine on the linker. Cleavage of photolabile linkers is usuallyperformed with UV light of 350 nm wavelength at intensities and timesknown to those in the art. Examples of conmmercial sources ofinstruments for photochemical cleavage are Aura Industries Inc. (StatenIsland, N.Y. and Agrenetics (Wilmington, Mass.). Cleavage of the linkersresults in liberation of a primary amide on the tag. Examples ofphotocleavable linkers include nitrophenyl glycine esters, exo- andendo-2-benzonorbomeyl chlorides and methane sulfonates, and3-amino-3(2-nitrophenyl) propionic acid. Examples of enzymatic cleavageinclude esterases which will cleave ester bonds, nucleases which willcleave phosphodiester bonds, proteases which cleave peptide bonds, etc.

E. Detection of Tags

Detection methods typically rely on the absorption and emission in sometype of spectral field. When atoms or molecules absorb light, theincoming energy excites a quantized structure to a higher energy level.The type of excitation depends on the wavelength of the light. Electronsare promoted to higher orbitals by ultraviolet or visible light,molecular vibrations are excited by infrared light, and rotations areexcited by microwaves. An absorption spectrum is the absorption of lightas a finction of wavelength. The spectrum of an atom or molecule dependson its energy level structure. Absorption spectra are usefuil foridentification of compounds. Specific absorption spectroscopic methodsinclude atomic absorption spectroscopy (AA), infrared spectroscopy (IR),and UV-vis spectroscopy (uv-vis).

Atoms or molecules that are excited to high energy levels can decay tolower levels by emitting radiation. This light emission is calledfluorescence if the transition is between states of the same spin, andphosphorescence if the transition occurs between states of differentspin. The emission intensity of an analyte is linearly proportional toconcentration (at low concentrations), and is useful for quantifying theemitting species. Specific emission spectroscopic methods include atomicemission spectroscopy (AES), atomic fluorescence spectroscopy (AFS),molecular laser-induced fluorescence (LIF), and X-ray fluorescence(XRF).

When electromagnetic radiation passes through matter, most of theradiation continues in its original direction but a small fraction isscattered in other directions. Light that is scattered at the samewavelength as the incoming light is called Rayleigh scattering. Lightthat is scattered in transparent solids due to vibrations (phonons) iscalled Brillouin scattering. Brillouin scattering is typically shiftedby 0.1 to 1 wave number from the incident light. Light that is scattereddue to vibrations in molecules or optical phonons in opaque solids iscalled Raman scattering. Raman scattered light is shifted by as much as4000 wavenumbers from the incident light. Specific scatteringspectroscopic methods include Raman spectroscopy.

IR spectroscopy is the measurement of the wavelength and intensity ofthe absorption of mid-infrared light by a sample. Mid-infrared light(2.5-50 μm, 4000-200 cm⁻¹) is energetic enough to excite molecularvibrations to higher energy levels. The wavelength of IR absorptionbands are characteristic of specific types of chemical bonds and IRspectroscopy is generally most useful for identification of organic andorganometallic molecules.

Near-infrared absorption spectroscopy (NIR) is the measurement of thewavelength and intensity of the absorption of near-infrared light by asample. Near-infrared light spans the 800 nm-2.5 μm (12,500-4000 cm⁻¹)range and is energetic enough to excite overtones and combinations ofmolecular vibrations to higher energy levels. NIR spectroscopy istypically used fot quantitative measurement of organic functionalgroups, especially O—H, N—H, and C═O. The components and design of NIRinstrumentation are similar to uv-vis absorption spectrometers. Thelight source is usually a tungsten lamp and the detector is usually aPbS solid-state detector. Sample holders can be glass or quartz andtypical solvents are CCl₄ and CS₂. The convenient instrumentation of NIRspectroscopy makes it suitable for on-line monitoring and processcontrol.

Ultraviolet and Visible Absorption Spectroscopy (uv-vis) spectroscopy isthe measurement of the wavelength and intensity of absorption ofnear-ultraviolet and visible light by a sample. Absorption in the vacuumUV occurs at 100-200 nm; (10⁵-50,000 cm⁻¹) quartz UV at 200-350 nm;(50,000-28,570 cm⁻¹) and visible at 350-800 nm; (28,570-12,500 cm⁻¹) andis described by the Beer-Lambert-Bouguet law. Ultraviolet and visiblelight are energetic enough to promote outer electrons to higher energylevels. UV-vis spectroscopy can be usually applied to molecules andinorganic ions or complexes in solution. The uv-vis spectra are limitedby the broad features of the spectra. The light source is usually ahydrogen or deuterium lamp for uv measurements and a tungsten lamp forvisible measurements. The wavelengths of these continuous light sourcesare selected with a wavelength separator such as a prism or gratingmonochromator. Spectra are obtained by scanning the wavelength separatorand quantitative measurements can be made from a spectrum or at a singlewavelength.

Mass spectrometers use the difference in the mass-to-charge ratio (m/z)of ionized atoms or molecules to separate them from each other. Massspectrometry is therefore useful for quantitation of atoms or moleculesand also for determining chemical and structural information aboutmolecules. Molecules have distinctive fragmentation patterns thatprovide structural information to identify compounds. The generaloperations of a mass spectrometer are as follows. Gas-phase ions arecreated, the ions are separated in space or time based on theirmass-to-charge ratio, and the quantity of ions of each mass-to-chargeratio is measured. The ion separation power of a mass spectrometer isdescribed by the resolution, which is defined as R=m/delta m, where m isthe ion mass and delta m is the difference in mass between tworesolvable peaks in a mass spectrum. For example, a mass spectrometerwith a resolution of 1000 can resolve an ion with a m/z of 100.0 from anion with a m/z of 100.1.

In general, a mass spectrometer (MS) consists of an ion source, amass-selective analyzer, and an ion detector. The magnetic-sector,quadrupole, and time-of-flight designs also require extraction andacceleration ion optics to transfer ions from the source region into themass analyzer. The details of several mass analyzer designs (formagnetic-sector MS, quadrupole MS or time-of-flight MS) are discussedbelow. Single Focusing analyzers for magnetic-sector MS utilize aparticle beam path of 180, 90, or 60 degrees. The various forcesinfluencing the particle separate ions with different mass-to-chargeratios. With double-focusing analyzers, an electrostatic analyzer isadded in this type of instrument to separate particles with differencein kinetic energies.

A quadrupole mass filter for quadrupole MS consists of four metal rodsarranged in parallel. The applied voltages affect the trajectory of ionstraveling down the flight path centered between the four rods. For givenDC and AC voltages, only ions of a certain mass-to-charge ratio passthrough the quadrupole filter and all other ions are thrown out of theiroriginal path. A mass spectrum is obtained by monitoring the ionspassing through the quadrupole filter as the voltages on the rods arevaried.

A time-of-flight mass spectrometer uses the differences in transit timethrough a “drift region” to separate ions of different masses. Itoperates in a pulsed mode so ions must be produced in pulses and/orextracted in pulses. A pulsed electric field accelerates all ions into afield-free drift region with a kinetic energy of qV, where q is the ioncharge and V is the applied voltage. Since the ion kinetic energy is 0.5mV², lighter ions have a higher velocity than heavier ions and reach thedetector at the end of the drift region sooner. The output of an iondetector is displayed on an oscilloscope as a function of time toproduce the mass spectrum.

The ion formation process is the starting point for mass spectrometricanalyses. Chemical ionization is a method that employs a reagent ion toreact with the analyte molecules (tags) to form ions by either a protonor hydride transfer. The reagent ions are produced by introducing alarge excess of methane (relative to the tag) into an electron impact(EI) ion source. Electron collisions produce CH₄ ⁺ and CH₃ ⁺ whichfurther react with methane to form CH₅ ⁺ and C₂H₅ ⁺. Another method toionize tags is by plasma and glow discharge. Plasma is a hot,partially-ionized gas that effectively excites and ionizes atoms. A glowdischarge is a low-pressure plasma maintained between two electrodes.Electron impact ionization employs an electron beam, usually generatedfrom a tungsten filament, to ionize gas-phase atoms or molecules. Anelectron from the beam knocks an electron off analyte atoms or moleculesto create ions. Electrospray ionization utilizes a very fine needle anda series of skimmers. A sample solution is sprayed into the sourcechamber to form droplets. The droplets carry charge when the exit thecapillary and as the solvent vaporizes the droplets disappear leavinghighly charged analyte molecules. ESI is particularly useful for largebiological molecules that are difficult to vaporize or ionize. Fast-atombombardment (FAB) utilizes a high-energy beam of neutral atoms,typically Xe or Ar, that strikes a solid sample causing desorption andionization. It is used for large biological molecules that are difficultto get into the gas phase. FAB causes little fragmentation and usuallygives a large molecular ion peak, making it useful for molecular weightdetermination. The atomic beam is produced by accelerating ions from anion source though a charge-exchange cell. The ions pick up an electronin collisions with neutral atoms to form a beam of high energy atoms.Laser ionization (LIMS) is a method in which a laser pulse ablatesmaterial from the surface of a sample and creates a microplasma thationizes some of the sample constituents. Matrix-assisted laserdesorption ionization (MALDI) is a LIMS method of vaporizing andionizing large biological molecules such as proteins or DNA fragments.The biological molecules are dispersed in a solid matrix such asnicotinic acid. A UV laser pulse ablates the matrix which carries someof the large molecules into the gas phase in an ionized form so they canbe extracted into a mass spectrometer. Plasma-desorption ionization (PD)utilizes the decay of ²⁵²Cf which produces two fission fragments thattravel in opposite directions. One fragment strikes the sample knockingout 1-10 analyte ions. The other fragment strikes a detector andtriggers the start of data acquisition. This ionization method isespecially useful for large biological molecules. Resonance ionization(RIMS) is a method in which one or more laser beams are tuned inresonance to transitions of a gas-phase atom or molecule to promote itin a stepwise fashion above its ionization potential to create an ion.Secondary ionization (SIMS) utilizes an ion beam; such as ³He⁺, ¹⁶O⁺, or⁴⁰Ar⁺; is focused onto the surface of a sample and sputters materialinto the gas phase. Spark source is a method which ionizes analytes insolid samples by pulsing an electric current across two electrodes.

A tag may become charged prior to, during or after cleavage from themolecule to which it is attached. Ionization methods based on ion“desorption”, the direct formation or emission of ions from solid orliquid surfaces have allowed increasing application to nonvolatile andthermally labile compounds. These methods eliminate the need for neutralmolecule volatilization prior to ionization and generally minimizethermal degradation of the molecular species. These methods includefield desorption (Becky, Principles of Field Ionization and FieldDesorption Mass Spectrometry, Pergamon, Oxford, 1977), plasma desorption(Sundqvist and Macfarlane, Mass Spectrom. Rev. 4:421, 1985), laserdesorption (Karas and Hillenkamp, Anal. Chem. 60:2299, 1988; Karas etal., Angew. Chem. 101:805, 1989), fast particle bombardment (e.g., fastatom bombardment, FAB, and secondary ion mass spectrometry, SIMS, Barberet al., Anal. Chem. 54:645A, 1982), and thermospray (TS) ionization(Vestal, Mass Spectrom. Rev. 2:447, 1983). Thermospray is broadlyapplied for the on-line combination with liquid chromatography. Thecontinuous flow FAB methods (Caprioli et al., Anal. Chem. 58:2949, 1986)have also shown significant potential. A more complete listing ofionization/mass spectrometry combinations is ion-trap mass spectrometry,electrospray ionization mass spectrometry, ion-spray mass spectrometry,liquid ionization mass spectrometry, atmospheric pressure ionizationmass spectrometry, electron ionization mass spectrometry, metastableatom bombardment ionization mass spectrometry, fast atom bombardionization mass spectrometry, MALDI mass spectrometry, photo-ionizationtime-of-flight mass spectrometry, laser droplet mass spectrometry,MALDI-TOF mass spectrometry, APCI mass spectrometry, nano-spray massspectrometry, nebulised spray ionization mass spectrometry, chemicalionization mass spectrometry., resonance ionization mass spectrometry,secondary ionization mass spectrometry, thermospray mass spectrometry.

The ionization methods amenable to nonvolatile biological compounds haveoverlapping ranges of applicability. Ionization efficiencies are highlydependent on matrix composition and compound type. Currently availableresults indicate that the upper molecular mass for T^(ms) is about 8000daltons (Jones and Krolik, Rapid Comm. Mass Spectrom. 1:67, 1987). SinceT^(ms) is practiced mainly with quadrapole mass spectrometers,sensitivity typically suffers disproportionately at highermass-to-charge ratios (m/z). Time-of-flight (TOF) mass spectrometers arecommercially available and possess the advantage that the m/z range islimited only by detector efficiency. Recently, two additional ionizationmethods have been introduced. These two methods are now referred to asmatrix-assisted laser desorption (MALDI, Karas and Hillenkamp, Anal.Chem. 60:2299, 1988; Karas et al., Angew. Chem. 101:805, 1989) andelectrospray ionization (ESI). Both methodologies have very highionization efficiency (i.e., very high [molecular ionsproduced]/[molecules consumed]). Sensitivity, which defines the ultimatepotential of the technique, is dependent on sample size, quantity ofions, flow rate, detection efficiency and actual ionization efficiency.

Electrospray-MS is based on an idea first proposed in the 1960s (Dole etal., J. Chem. Phys. 49:2240, 1968). Electrospray ionization (ESI) is onemeans to produce charged molecules for analysis by mass spectroscopy.Briefly, electrospray ionization produces highly charged droplets bynebulizing liquids in a strong electrostatic field. The highly chargeddroplets, generally formed in a dry bath gas at atmospheric pressure,shrink by evaporation of neutral solvent until the charge repulsionovercomes the cohesive forces, leading to a “Coulombic explosion”. Theexact mechanism of ionization is controversial and several groups haveput forth hypotheses (Blades et al., Anal. Chem. 63:2109-14, 1991;Kebarle et al., Anal. Chem. 65:A972-86, 1993; Fenn, J. Am. Soc. Mass.Spectrom. 4:524-35, 1993). Regardless of the ultimate process of ionformation, ESI produces charged molecules from solution under mildconditions.

The ability to obtain useful mass spectral data on small amounts of anorganic molecule relies on the efficient production of ions. Theefficiency of ionization for ESI is related to the extent of positivecharge associated with the molecule. Improving ionization experimentallyhas usually involved using acidic conditions. Another method to improveionization has been to use quaternary amines when possible (seeAebersold et al., Protein Science 1:494-503, 1992; Smith et al., Anal.Chem. 60:436-41, 1988).

Electrospray ionization is described in more detail as follows.Electrospray ion production requires two steps: dispersal of highlycharged droplets at near atmospheric pressure, followed by conditions toinduce evaporation. A solution of analyte molecules is passed through aneedle that is kept at high electric potential. At the end of theneedle, the solution disperses into a mist of small highly chargeddroplets containing the analyte molecules. The small droplets evaporatequickly and by a process of field desorption or residual evaporation,protonated protein molecules are released into the gas phase. Anelectrospray is generally produced by application of a high electricfield to a small flow of liquid (generally 1-10 uL/min) from a capillarytube. A potential difference of 3-6 kV is typically applied between thecapillary and counter electrode located 0.2-2 cm away (where ions,charged clusters, and even charged droplets, depending on the extent ofdesolvation, may be sampled by the MS through a small orifice). Theelectric field results in charge accumulation on the liquid surface atthe capillary terminus; thus the liquid flow rate, resistivity, andsurface tension are important factors in droplet production. The highelectric field results in disruption of the liquid surface and formationof highly charged liquid droplets. Positively or negatively chargeddroplets can be produced depending upon the capillary bias. The negativeion mode requires the presence of an electron scavenger such as oxygento inhibit electrical discharge.

A wide range of liquids can be sprayed electrostatically into a vacuum,or with the aid of a nebulizing agent. The use of only electric fieldsfor nebulization leads to some practical restrictions on the range ofliquid conductivity and dielectric constant. Solution conductivity ofless than 10⁻⁵ ohms is required at room temperature for a stableelectrospray at useful liquid flow rates corresponding to an aqueouselectrolyte solution of <10⁻⁴ M. In the mode found most useful forESI-MS, an appropriate liquid flow rate results in dispersion of theliquid as a fine mist. A short distance from the capillary the dropletdiameter is often quite uniform and on the order of 1 μm. Of particularimportance is that the total electrospray ion current increases onlyslightly for higher liquid flow rates. There is evidence that heating isuseful for manipulating the electrospray. For example, slight heatingallows aqueous solutions to be readily electrosprayed, presumably due tothe decreased viscosity and surface tension. Both thermally-assisted andgas-nebulization-assisted electrosprays allow higher liquid flow ratesto be used, but decrease the extent of droplet charging. The formationof molecular ions requires conditions effecting evaporation of theinitial droplet population. This can be accomplished at higher pressuresby a flow of dry gas at moderate temperatures (<60° C.), by heatingduring transport through the interface, and (particularly in the case ofion trapping methods) by energetic collisions at relatively lowpressure.

Although the detailed processes underlying ESI remain uncertain, thevery small droplets produced by ESI appear to allow almost any speciescarrying a net charge in solution to be transferred to the gas phaseafter evaporation of residual solvent. Mass spectrometric detection thenrequires that ions have a tractable m/z range (<4000 daltons forquadrupole instruments) after desolvation, as well as to be produced andtransmitted with sufficient efficiency. The wide range of solutesalready found to be amenable to ESI-MS, and the lack of substantialdependence of ionization efficiency upon molecular weight, suggest ahighly non-discriminating and broadly applicable ionization process.

The electrospray ion “source” functions at near atmospheric pressure.The electrospray “source” is typically a metal or glass capillaryincorporating a method for electrically biasing the liquid solutionrelative to a counter electrode. Solutions, typically water-methanolmixtures containing the analyte and often other additives such as aceticacid, flow to the capillary terminus. An ESI source has been described(Smith et al., Anal. Chem. 62:885, 1990) which can accommodateessentially any solvent system. Typical flow rates for ESI are 1-10uL/min. The principal requirement of an ESI-MS interface is to sampleand transport ions from the high pressure region into the MS asefficiently as possible.

The efficiency of ESI can be very high, providing the basis forextremely sensitive measurements, which is useful for the inventiondescribed herein. Current instrumental performance can provide a totalion current at the detector of about 2×10⁻¹² A or about 10⁷ counts/s forsingly charged species. On the basis of the instrumental performance,concentrations of as low as 10⁻¹⁰ M or about 10⁻¹⁸ mol/s of a singlycharged species will give detectable ion current (about 10 counts/s) ifthe analyte is completely ionized. For example, low attomole detectionlimits have been obtained for quaternary ammonium ions using an ESIinterface with capillary zone electrophoresis (Smith et al., Anal. Chem.59:1230, 1988). For a compound of molecular weight of 1000, the averagenumber of charges is 1, the approximate number of charge states is 1,peak width (m/z) is 1 and the maximum intensity (ion/s) is 1×10¹².

Remarkably little sample is actually consumed in obtaining an ESI massspectrum (Smith et al., Anal. Chem. 60:1948, 1988). Substantial gainsmight be also obtained by the use of array detectors with sectorinstruments, allowing simultaneous detection of portions of thespectrum. Since currently only about 10⁻⁵ of all ions formed by ESI aredetected, attention to the factors limiting instrument performance mayprovide a basis for improved sensitivity. It will be evident to those inthe art that the present invention contemplates and accommodates forimprovements in ionization and detection methodologies.

An interface is preferably placed between the separation instrumentation(e.g., gel) and the detector (e.g., mass spectrometer). The interfacepreferably has the following properties: (1) the ability to collect theDNA fragments at discreet time intervals, (2) concentrate the DNAfragments, (3) remove the DNA fragments from the electrophoresis buffersand milieu, (4) cleave the tag from the DNA fragment, (5) separate thetag from the DNA fragment, (6) dispose of the DNA fragment, (7) placethe tag in a volatile solution, (8) volatilize and ionize the tag, and(9) place or transport the tag to an electrospray device that introducesthe tag into mass spectrometer.

The interface also has the capability of “collecting” DNA fragments asthey elute from the bottom of a gel. The gel may be composed of a slabgel, a tubular gel, a capillary, etc. The DNA fragments can be collectedby several methods. The first method is that of use of an electric fieldwherein DNA fragments are collected onto or near an electrode. A secondmethod is that wherein the DNA fragments are collected by flowing astream of liquid past the bottom of a gel. Aspects of both methods canbe combined wherein DNA collected into a flowing stream which can belater concentrated by use of an electric field. The end result is thatDNA fragments are removed from the milieu under which the separationmethod was performed. That is, DNA fragments can be “dragged” from onesolution type to another by use of an electric field.

Once the DNA fragments are in the appropriate solution (compatible withelectrospray and mass spectrometry) the tag can be cleaved from the DNAfragment. The DNA fragment (or remnants thereof) can then be separatedfrom the tag by the application of an electric field (preferably, thetag is of opposite charge of that of the DNA tag). The tag is thenintroduced into the clectrospray device by the use of an electric fieldor a flowing liquid.

Fluorescent tags can be identified and quantitated most directly bytheir absorption and fluorescence emission wavelengths and intensities.

While a conventional spectrofluorometer is extremely flexible, providingcontinuous ranges of excitation and emission wavelengths (l_(EX),l_(S1), l_(S2)), more specialized instruments such as flow cytometersand laser-scanning microscopes require probes that are excitable at asingle fixed wavelength. In contemporary instruments, this is usuallythe 488-nm line of the argon laser.

Fluorescence intensity per probe molecule is proportional to the productof e and QY. The range of these parameters among fluorophores of currentpractical importance is approximately 10,000 to 100,000 cm⁻¹M⁻¹ for εand 0.1 to 1.0 for QY. When absorption is driven toward saturation byhigh-intensity illumination, the irreversible destruction of the excitedfluorophore (photobleaching) becomes the factor limiting fluorescencedetectability. The practical impact of photobleaching depends on thefluorescent detection technique in question.

It will be evident to one in the art that a device (an interface) may beinterposed between the separation and detection steps to permit thecontinuous operation of size separation and tag detection (in realtime). This unites the separation methodology and instrumentation withthe detection methodology and instrumentation forming a single device.For example, an interface is interposed between a separation techniqueand detection by mass spectrometry or potentiostatic amperometry.

The function of the interface is primarily the release of the (e.g.,mass spectrometry) tag from analyte. There are several representativeimplementations of the interface. The design of the interface isdependent on the choice of cleavable linkers. In the case of light orphoto-cleavable linkers, an energy or photon source is required. In thecase of an acid-labile linker, a base-labile linker, or a disulfidelinker, reagent addition is required within the interface. In the caseof heat-labile linkers, an energy heat source is required. Enzymeaddition is required for an enzyme-sensitive linker such as a specificprotease and a peptide linker, a nuclease and a DNA or RNA linker, aglycosylase, HRP or phosphatase and a linker which is unstable aftercleavage (e.g., similar to chemiluminescent substrates). Othercharacteristics of the interface include minimal band broadening,separation of DNA from tags before injection into a mass spectrometer.Separation techniques include those based on electrophoretic methods andtechniques, affinity techniques, size retention (dialysis), filtrationand the like.

It is also possible to concentrate the tags (or nucleic acid-linker-tagconstruct), capture electrophoretically, and then release into alternatereagent stream which is compatible with the particular type ofionization method selected. The interface may also be capable ofcapturing the tags (or nucleic acid-linker-tag construct) on microbeads,shooting the bead(s) into chamber and then performing laserdesorption/vaporization. Also it is possible to extract in flow intoalternate buffer (e.g., from capillary electrophoresis buffer intohydrophobic buffer across a permeable membrane). It may also bedesirable in some uses to deliver tags into the mass spectrometerintermittently which would comprise a further function of the interface.Another function of the interface is to deliver tags from multiplecolumns into a mass spectrometer, with a rotating time slot for eachcolumn. Also. it is possible to deliver tags from a single column intomultiple MS detectors, separated by time, collect each set of tags for afew milliseconds, and then deliver to a mass spectrometer.

The following is a list of representative vendors for separation anddetection technologies which may be used in the present invention.Hoefer Scientific Instruments (San Francisco, Calif.) manufactureselectrophoresis equipment (Two Step™, Poker Face™ II) for sequencingapplications. Pharmacia Biotech (Piscataway, N.J.) manufactureselectrophoresis equipment for DNA separations and sequencing(PhastSystem for PCR-SSCP analysis, MacroPhor System for DNAsequencing). Perkin Elmer/Applied Biosystems Division (ABI, Foster City,Calif.) manufactures semi-automated sequencers based on fluorescent-dyes(ABI373 and ABI377). Analytical Spectral Devices (Boulder, Colo.)manufactures UV spectrometers. Hitachi Instruments (Tokyo, Japan)manufactures Atomic Absorption spectrometers, Fluorescencespectrometers, LC and GC Mass Spectrometers, NMR spectrometers, andUV-VIS Spectrometers. PerSeptive Biosystems (Framingham, Mass.) producesMass Spectrometers (Voyager™ Elite). Bruker Instruments Inc. (ManningPark, Mass.) manufactures FTIR Spectrometers (Vector 22), FT-RamanSpectrometers, Time of Flight Mass Spectrometers (Reflex II™), Ion TrapMass Spectrometer (Esquire™) and a Maldi Mass Spectrometer. AnalyticalTechnology Inc. (ATI, Boston, Mass.) makes Capillary Gel Electrophoresisunits, UV detectors, and Diode Array Detectors. Teledyne ElectronicTechnologies (Mountain View, Calif.) manufactures an Ion Trap MassSpectrometer (3DQ Discovery™ and the 3DQ Apogee™). Perkin Elmer/AppliedBiosystems Division (Foster City, Calif.) manufactures a Sciex MassSpectrometer (triple quadrupole LC/MS/MS, the API 100/300) which iscompatible with electrospray. Hewlett-Packard (Santa Clara, Calif.)produces Mass Selective Detectors (HP 5972A), MALDI-TOF MassSpectrometers (HP G2025A), Diode Array Detectors, CE units, HPLC units(HP1090) as well as UV Spectrometers. Finnigan Corporation (San Jose,Calif.) manufactures mass spectrometers (magnetic sector (MAT 95 S™),quadrapole spectrometers (MAT 95 SQ™) and four other related massspectrometers). Rainin (Emeryville, Calif.) manufactures HPLCinstruments.

The methods and compositions described herein permit the use of cleavedtags to serve as maps to particular sample type and nucleotide identity.At the beginning of each sequencing method, a particular (selected)primer is assigned a particular unique tag. The tags map to either asample type, a dideoxy terminator type (in the case of a Sangersequencing reaction) or preferably both. Specifically, the tag maps to aprimer type which in turn maps to a vector type which in turn maps to asample identity. The tag may also may map to a dideoxy terminator type(ddTTP, ddCTP, ddGTP, ddATP) by reference into which dideoxynucleotidereaction the tagged primer is placed. The sequencing reaction is thenperformed and the resulting fragments are sequentially separated by sizein time.

The tags are cleaved from the fragments in a temporal frame and measuredand recorded in a temporal frame. The sequence is constructed bycomparing the tag map to the temporal frame. That is, all tag identitiesare recorded in time after the sizing step and related become related toone another in a temporal frame. The sizing step separates the nucleicacid fragments by a one nucleotide increment and hence the related tagidentities are separated by a one nucleotide increment. By foreknowledgeof the dideoxy-terminator or nucleotide map and sample type, thesequence is readily deduced in a linear fashion.

A DNA sequencing system of an exemplary embodiment of the presentinvention consists of, in general, a sample introduction device, adevice to separate the tagged samples of interest, a device to cleavethe tags from the samples of interest, a device for detecting the tag,and a software program to analyze the data collected. It will be evidentto one of ordinary skill in the art when in possession of the presentdisclosure that this general description may have many variances foreach of the components listed. As best seen in FIG. 15, an exemplaryembodiment of the DNA sequencing system 10 of the present inventionconsists of a sample introduction device 12, a separation device 14 thatseparates the samples by high-performance liquid chromatography (HPLC),a photocleavage device 16 to cleave the tags from the samples ofinterest, a detection device 18 that detects the tags by massspectrometry, and a data processing device 20 with a data analysissoftware program that analyzes the results from the detection device 16.Each component is discussed in more detail below.

The sample introduction device 12 automatically takes a measured aliquot22 (e.g., of the PCR product generated by the Sanger sequencing method)and delivers it through a conventional tube 24 to the separation device14 (generally an HPLC). The sample introduction device 12 of theexemplary embodiment consists of a temperature-controlled autosampler 26that can accommodate micro-titer plates. The autosampler 26 must betemperature controlled to maintain the integrity of the nucleic acidsamples generated and be able to inject 25 μl or less of sample.Manufacturers of this type of sample introduction device 12 arerepresented, for example, by Gilson (Middleton, Wis.).

The sample introduction device 12 is operatively connected in series tothe separation device 14 by the tube 24. The sequencing reactionproducts (which may be produced by PCR) in the measured aliquot 22 arereceived in the separation device 14 and separated temporally byhigh-performance liquid chromatography to provide separated DNAfragments. The high-performance liquid chromatograph may have anisocratic, binary, or quaternary pump(s) 27 and can be purchased frommultiple manufacturers (e.g., Hewlett Packard (Palo Alto, Calif.) HP1100 or 1090 series, Beckman Instruments Inc. (800-742-2345),Bioanalytical Systems, Inc. (800-845-4246), ESA, Inc. (508) 250-700),Perkin-Elmer Corp. (800-762-4000), Varian Instruments (800-926-3000),Waters Corp. (800-254-4752)).

The separation device 14 includes an analytical HPLC column 28 suitablefor use to separate the nucleic acid fragments. The column 28 is ananalytical HPLC, for example, is non-porous polystyrene divinylbenzene(2.2μ particle size) solid support which can operate within a pH rangeof 2 to 12, pressures of up to 3000 psi and a temperature range of about10 to 70° C. A temperature-control device (e.g., a column oven) (notshown) may be used to control the temperature of the column. Suchtemperature-control devices are known in the art, and may be obtainedfrom, for example, Rainin Instruments (subsidiary of Varian Instruments,Palo Alto, Calif.). A suitable column 28 is available under thecommercial name of DNAsep® and is available from Serasep (San Jose,Calif.). Other suitable analytical HPLC columns are available from othermanufacturers (e.g., Hewlett Packard (Palo Alto, Calif.), BeckmanInstruments Inc. (Brea, Calif.), Waters Corp. (Milford, Mass.)).

A stream of the separated DNA fragments (e.g., sequencing reactionproducts) flows through a conventional tube 30 from the separationdevice 14 to the cleavage device 16. Each of the DNA fragments islabeled with a unique cleavable (e.g., photocleavable) tag. The flowingstream of separated DNA fragments pass through or past the cleavingdevice 16 where the tag is removed for detection (e.g., by massspectrometry or with an electrochemical detector). In the exemplaryembodiment, the cleaving device 16 is a photocleaving unit such that theflowing stream of sample is exposed to selected light energy andwavelength. In one embodiment, the sample enters the photocleaving unitand is exposed to the selected light source for a selected duration oftime. In an alternate embodiment, the stream of separated DNA fragmentsis carried adjacent to the light source along a path that provides asufficient exposure to the light energy to cleave the tags from theseparated DNA fragments.

A photocleaving unit is available from Supelco (Bellefonte, Pa.).Photocleaving can be performed at multiple wavelengths with amercury/xenon arc lamp. The wavelength accuracy is about 2 nm with abandwidth of 10 nm. The area irradiated is circular and typically of anarea of 10-100 square centimeters. In alternate embodiments, othercleaving devices, which cleave by acid, base, oxidation, reduction,floride, thiol exchange, photolysis, or enzymatic conditions, can beused to remove the tags from the separated DNA fragments.

After the cleaving device 16 cleaves the tags from the separated DNAfragments, the tags flow through a conventional tube 32 to the detectiondevice 18 for detection of each tag. Detection of the tags can be basedupon the difference in molecular weight between each of the tags used tolabel each kind of DNA generated in the PCR step. The best detectorbased upon differences in mass is the mass spectrometer. For this use,the mass spectrometer typically will have an atmospheric pressureionization (API) interface with either electrospray or chemicalionization, a quadrupole mass analyzer, and a mass range of at least 50to 2600 m/z. Examples of manufacturers of a suitable mass spectrometerare: Hewlett Packard (Palo Alto, Calif.) HP 1100 LC/MSD, HitachiInstruments (San Jose, Calif.) M-1200H LC/MS, Perkin Elmer Corporation,Applied Biosystems Division (Foster City, Calif.) API 100 LC/MS or API300 LC/MS/MS, Finnigan Corporation (San Jose, Calif.) LCQ, BrukerAnalytical Systems, Inc. (Billerica, Mass.) ESQUIRE and MicroMass (UK).

The detection device 18 is electrically connected to a data processorand analyzer 20 that receives data from the detection device. The dataprocessor and analyzer 20 includes a software program that identifiesthe detected tag. The data processor and analyzer 20 in alternateembodiments is operatively connected to the sample introduction device12, the separation device 14, and/or the cleaving device 16 to controlthe different components of the DNA sequencing system 10.

In an alternate embodiment illustrated schematically in FIG. 16, thesample introduction device 12, the separating device 14, and thecleavage device 16 are serially connected as discussed above formaintaining the flow of the sample. The cleavage device 16 is seriallyconnected to detection device 18 that is an electrochemical detectingdevice 40 rather than a mass spectrometer. Detection of the tags in thisalternate embodiment is based upon the difference in electrochemicalpotential between each of the tags used to label each kind of DNAgenerated in the sequencing reaction step. The electrochemical detector40 can operate on either coulometric or amperometric principles. Thepreferred electrochemical detector 40 is the coulometric detector, whichconsists of a flow-through or porous-carbon graphite amperometricdetector where the column eluent passes through the electrode resultingin 100% detection efficiency. To fully detect each component, an arrayof 16 coulometric detectors each held at a different potential(generally at 60 mV increments) is utilized. Examples of manufacturersof this type of detector are ESA (Bedford, Mass.) and BioanalyticalSystems Inc. (800-845-4246).

The electrochemical detector 40 is electrically connected to the dataprocessor and analyzer 20 with the software package. The softwarepackage maps the detected property (e.g., the electrochemical signatureor mass as discussed above) of a given tag to a specific sample ID. Thesoftware is able to display the DNA sequence determined and load thesequence information into respective databases.

The DNA sequencing system 10 is provided by operatively interconnectingthe system's multiple components. Accordingly, one or more systemcomponents, such as the sample introducing device 12 and the detectingdevice 18 that are in operation in a lab can be combined with thesystem's other components (e.g., the separating device 14, cleavingdevice 16, and the data processor and analyzer 20 in order to equip thelab with the DNA sequencing system 10 of the present invention.

In a preferred embodiment, five or more (and more preferably sixteen ormore) samples are introduced simultaneously in a system according to thepresent invention. Each sample has a unique tag for each type ofnucleotide and the system has a data processor device that correlatesthe detection of a tag (e.g., tag mass or tag electrochemical signature)to a particular nucleotide and to a specific sample. For example, wherefive samples are introduced into the system, the data processor is ableto associate a tag detected by the system with both the particularnucleotide (to which the tag was attached prior to cleavage) and one ofthe five specific samples (to which the tag was introduced to generatetagged nucleic acid fragments).

Tagged Probes in Array-Based Assays

Arrays with covalently attached oligonucleotides have been made used toperform DNA sequence analysis by hybridization (Southern et al.,Genomics 13: 1008, 1992; Drmanac et al., Science 260: 1649, 1993),determine expression profiles, screen for mutations and the like. Ingeneral, detection for these assays uses fluorescent or radioactivelabels. Fluorescent labels can be identified and quantitated mostdirectly by their absorption and fluorescence emission wavelengths andintensity. A microscope/camera setup using a fluorescent light source isa convenient means for detecting fluorescent label. Radioactive labelsmay be visualized by standard autoradiography, phosphor image analysisor CCD detector. For such labels the number of different reactions thatcan be detected at a single time is limited. For example, the use offour fluorescent molecules, such as commonly employed in DNA sequenceanalysis, limits anaylsis to four samples at a time. Essentially,because of this limitation, each reaction must be individually assessedwhen using these detector methods.

A more advantageous method of detection allows pooling of the samplereactions on at least one array and simultaneous detection of theproducts. By using a tag, such as the ones described herein, having adifferent molecular weight or other physical attribute in each reaction,the entire set of reaction products can be harvested together andanalyzed.

As noted above, the methods described herein are applicable for avariety of purposes. For example, the arrays of oligonucleotides may beused to control for quality of making arrays, for quantitation orqualitative analysis of nucleic acid molecules, for detecting mutations,for determining expression profiles, for toxicology testing, and thelike.

Probe quantitation or typing

In this embodiment, oligonucleotides are immobilized per element in anarray where each oligonucleotide in the element is a different orrelated sequence. Preferably, each element possesses a known or relatedset of sequences. The hybridization of a labeled probe to such an arraypermits the characterization of a probe and the identification andquantification of the sequences contained in a probe population.

A generalized assay format that may be used in the particularapplications discussed below is a sandwich assay format. In this format,a plurality of oligonucleotides of known sequence are immobilized on asolid substrate. The immobilized oligonucleotide is used to capture anucleic acid (e.g., RNA, rRNA, a PCR product, fragmented DNA) and then asignal probe is hybridized to a different portion of the captured targetnucleic acid.

Another generalized assay format is a secondary detection system. Inthis format, the arrays are used to identify and quantify labelednucleic acids that have been used in a primary binding assay. Forexample, if an assay results in a labeled nucleic acid, the identity ofthat nucleic acid can be determined by hybridization to an array. Theseassay formats are particularly useful when combined with cleavable massspectometry tags.

Mutation detection

Mutations involving a single nucleotide can be identified in a sample byscanning techniques, which are suitable to identify previously unknownmutations, or by techniques designed to detect, distinguish, orquantitate known sequence variants. Several scanning techniques formutation detection have been developed based on the observation thatheteroduplexes of mismatched complementary DNA strands, derived fromwild type and mutant sequences, exhibit an abnormal migratory behavior.

The methods described herein may be used for mutation screening. Onestrategy for detecting a mutation in a DNA strand is by hybridization ofthe test sequence to target sequences that are wild-type or mutantsequences. A mismatched sequence has a destabilizing effect on thehybridization of short oligonucleotide probes to a target sequence (seeWetmur, Crit. Rev. Biochem. Mol. Biol., 26:227, 1991). The test nucleicacid source can be genomic DNA, RNA, cDNA, or amplification of any ofthese nucleic acids. Preferably, amplification of test sequences isfirst performed, followed by hybridization with short oligonucleotideprobes immobilized on an array. An amplified product can be scanned formany possible sequence variants by determining its hybridization patternto an array of immobilized oligonucleotide probes.

A label, such as described herein, is generally incorporated into thefinal amplification product by using a labeled nucleotide or by using alabeled primer. The amplification product is denatured and hybridized tothe array. Unbound product is washed off and label bound to the array isdetected by one of the methods herein. For example, when cleavable massspectrometry tags are used, multiple products can be simultaneouslydetected.

Expression profiles/differential display

Mammals, such as human beings, have about 100,000 different genes intheir genome, of which only a small fraction, perhaps 15%, are expressedin any individual cell. Differential display techniques permit theidentification of genes specific for individual cell types. Briefly, indifferential display, the 3′terminal portions of mRNAs are amplified andidentified on the basis of size. Using a primer designed to bind to the5′ boundary of a poly(A) tail for reverse transcription, followed byamplification of the cDNA using upstream arbitrary sequence primers,mRNA sub-populations are obtained.

As disclosed herein, a high throughput method for measuring theexpression of numerous genes (e.g., 1-2000) is provided. Within oneembodiment of the invention, methods are provided for analyzing thepattern of gene expression from a selected biological sample, comprisingthe steps of (a) amplifying cDNA from a biological sample using one ormore tagged primers, wherein the tag is correlative with a particularnucleic acid probe and detectable by non-fluorescent spectrometry orpotentiometry, (b) hybridizing amplified fragments to an array ofoligonucleotides as described herein, (c) washing away non-hybridizedmaterial, and (d) detecting the tag by non-fluorescent spectrometry orpotentiometry, and therefrom determining the pattern of gene expressionof the biological sample. Tag-based differential display, especiallyusing cleavable mass spectometry tags, on solid substrates allowscharacterization of differentially expressed genes.

Single nucleotide extension assay

The primer extension technique may be used for the detection of singlenucleotide changes in a nucleic acid template (Sokolov, Nucleic AcidsRes., 18:3671, 1989). The technique is generally applicable to detectionof any single base mutation (Kuppuswamy et al., Proc. Natl, Acad. Sci.USA, 88:1143-1147, 1991). Briefly, this method first hybridizes a primerto a sequence adjacent to a known single nucleotide polymorphism. Theprimed DNA is then subjected to conditions in which a DNA polymeraseadds a labeled dNTP, typically a ddNTP, if the next base in the templateis complementary to the labeled nucleotide in the reaction mixture. In amodification, cDNA is first amplified for a sequence of interestcontaining a single-base difference between two alleles. Each amplifiedproduct is then analyzed for the presence, absence, or relative amountsof each allele by annealing a primer that is 1 base 5′ to thepolymorphism and extending by one labeled base (generally adideoxynucleotide). Only when the correct base is available in thereaction will a base to incorporated at the 3′-end of the primer.Extension products are then analyzed by hybridization to an array ofoligonucleotides such that a non-extended product will not hybridize.

Briefly, in the present invention, each dideoxynucleotide is labeledwith a unique tag. Of the four reaction mixtures, only one will add adideoxy-terminator on to the primer sequence. If the mutation ispresent, it will be detected through the unique tag on thedideoxynucleotide after hybridization to the array. Multiple mutationscan be simultaneously determined by tagging the DNA primer with a uniquetag as well. Thus, the DNA fragments are reacted in four separatereactions each including a different tagged dideoxyterminator, whereinthe tag is correlative with a particular dideoxynucleotide anddetectable by non-fluorescent spectrometry, or potentiometry. The DNAfragments are hybridized to an array and non-hybridized material iswashed away. The tags are cleaved from the hybridized fragments anddetected by the respective detection technology (e.g., massspectrometry, infrared spectrometry, potentiostatic amperometry orUV/visible spectrophotometry). The tags detected can be correlated tothe particular DNA fragment under investigation as well as the identityof the mutant nucleotide.

Oligonucleotide ligation assay

The oligonucleotide ligation assay (OLA). (Landegen et al.,Science241:487, 1988) is used for the identification of known sequencesin very large and complex genomes. The principle of OLA is based on theability of ligase to covalently join two diagnostic oligonucleotides asthey hybridize adjacent to one another on a given DNA target. If thesequences at the probe junctions are not perfectly based-paired, theprobes will not be joined by the ligase. When tags are used, they areattached to the probe, which is ligated to the amplified product. Aftercompletion of OLA, fragments are hybridized to an array of complementarysequences, the tags cleaved and detected by mass spectrometry.

Within one embodiment of the invention methods are provided fordetermining the identity of a nucleic acid molecule, or for detecting aselecting nucleic acid molecule, in, for example a biological sample,utilizing the technique of oligonucleotide ligation assay. Briefly, suchmethods generally comprise the steps of performing amplification on thetarget DNA followed by hybridization with the 5′ tagged reporter DNAprobe and a 5′ phosphorylated probe. The sample is incubated with T4 DNAligase. The DNA strands with ligated probes are captured on the array byhybridization to an array, wherein non-ligated products do nothybridize. The tags are cleaved from the separated fragments, and thenthe tags are detected by the respective detection technology (e.g., massspectrometry, infrared spectrophotometry, potentiostatic amperometry orUV/visible spectrophotometry.

Other assays

The methods described herein may also be used to genotype oridentification of viruses or microbes. For example, F+RNA coliphages maybe useful candidates as indicators for enteric virus contamination.Genotyping by nucleic acid amplification and hybridization methods arereliable, rapid, simple, and inexpensive alternatives to serotyping(Kafatos et. al., Nucleic Acids Res. 7:1541, 1979). Amplificationtechniques and nucleic aid hybridization techniques have beensuccessfully used to classify a variety of microorganisms including E.coli (Feng, Mol. Cell Probes 7:151, 1993), rotavirus (Sethabutr et. al.,J. Med Virol. 37:192, 1992), hepatitis C virus (Stuyver et. al., J. GenVirol. 74:1093, 1993), and herpes simplex virus (Matsumoto et. al., J.Virol. Methods 40:119, 1992).

Genetic alterations have been described in a variety of experimentalmammalian and human neoplasms and represent the morphological basis forthe sequence of morphological alterations observed in carcinogenesis(Vogelstein et al., NEJM319:525, 1988). In recent years with the adventof molecular biology techniques, allelic losses on certain chromosomesor mutation of tumor suppressor genes as well as mutations in severaloncogenes (e.g., c-myc, c-jun, and the ras family) have been the moststudied entities. Previous work (Finkelstein et al., Arch Surg. 128:526,1993) has identified a correlation between specific types of pointmutations in the K-ras oncogene and the stage at diagnosis in colorectalcarcinoma. The results suggested that mutational analysis could provideimportant information of tumor aggressiveness, including the pattern andspread of metastasis. The prognostic value of TP53 and K-ras-2mutational analysis in stage III carconoma of the colon has morerecently been demonstrated (Pricolo et al., Am. J. Surg. 171:41, 1996).It is therefore apparent that genotyping of tumors and pre-cancerouscells, and specific mutation detection will become increasinglyimportant in the treatment of cancers in humans.

The tagged biomolecules as disclosed herein may be used to interrogate(untagged) arrays of biomolecules. Preferred arrays of biomolculescontain a solid substrate comprising a surface, where the surface is atleast partially covered with a layer of poly(ethylenimine) (PEI). ThePEI layer comprises a plurality of discrete first regions abutted andsurrounded by a contiguous second region. The first regions are definedby the presence of a biomolecule and PEI, while the second region isdefined by the presence of PEI and the substantial absence of thebiomolecule. Preferably, the substrate is a glass plate or a siliconwafer. However, the substrate may be, for example, quartz, gold,nylon-6,6, nylon or polystyrene, as well as composites thereof, asdescribed above.

The PEI coating preferably contains PEI having a molecular weightranging from 100 to 100,000. The PEI coating may be directly bonded tothe substrate using, for example, silylated PEI. Alternatively, areaction product of a biftinctional coupling agent may be disposedbetween the substrate surface and the PEI coating, where the reactionproduct is covalently bonded to both the surface and the PEI coating,and secures the PEI coating to the surface. The bifunctional couplingagent contains a first and a second reactive functional group, where thefirst reactive functional group is, for example, atri(O—C₁-C₅alkyl)silane, and the second reactive functional group is,for example, an epoxide, isocyanate, isothiocyanate and anhydride group.Preferred bifuictional coupling agents include2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane;3,4-epoxybutyltrimethoxysilane; 3-isocyanatopropyltriethoxysilane,3-(triethoxysilyl)-2-methylpropylsuccinic anhydride and3-(2,3-epoxypropoxy)propyltrimethoxysilane.

The array of the invention contains first, biomolecule-containingregions, where each region has an area within the range of about 1,000square microns to about 100,000 square microns. In a preferredembodiment, the first regions have areas that range from about 5,000square microns to about 25,000 square microns.

The first regions are preferably substantially circular, where thecircles have an average diameter of about 10 microns to 200 microns.Whether circular or not, the boundaries of the first regions arepreferably separated from one another (by the second region) by anaverage distance of at least about 25 microns, however by not more thanabout 1 cm (and preferably by no more than about 1,000 microns). In apreferred array, the boundaries of neighboring first regions areseparated by an average distance of about 25 microns to 100 microns,where that distance is preferably constant throughout the array, and thefirst regions are preferably positioned in a repeating geometric patternas shown in the Figures attached hereto. In a preferred repeatinggeometric pattern, all neighboring first regions are separated byapproximately the same distance (about 25 microns to about 100 microns).

In preferred arrays, there are from 10 to 50 first regions on thesubstrate. In another embodiment, there are 50 to 400 first regions on asubstrate. In yet another preferred embodiment, there are 400 to 800first regions on the substrate.

The biomolecule located in the first regions is preferably a nucleicacid polymer. A preferred nucleic acid polymer is an oligonucleotidehaving from about 15 to about 50 nucleotides. The biomolecule may beamplification reaction products having from about 50 to about 1,000nucleotides.

In each first region, the biomolecule is preferably present at anaverage concentration ranging from 10⁵ to 10⁹ biomolecules per 2,000square microns of a first region. More preferably, the averageconcentration of biomolecule ranges from 10⁷ to 10⁹ biomolecules per2,000 square microns. In the second region, the biomolecule ispreferably present at an average concentration of less than 10³biomolecules per 2,000 square microns of said second region, and morepreferably at an average concentration of less than 10² biomolecules per2,000 square microns. Most preferably, the second regions does notcontain any biomolecule.

The chemistry used to adhere the layer of PEI to the substrate depends,in substantial part, upon the chemical identity of the substrate. Theprior art provides numerous examples of suitable chemistries that mayadhere PEI to a solid support. For example, when the substrate isnylon-6,6, the PEI coating may be applied by the methods disclosed inVan Ness, J. et al. Nucleic Acids Res. 19:3345-3350, 1991 and PCTInternational Publication WO 94/00600, both of which are incorporatedherein by reference. When the solid support is glass or silicon,suitable methods of applying a layer of PEI are found in, e.g.,Wasserman, B. P. Biotechnology and Bioengineering XXII:271-287, 1980;and D'Souza, S. F. Biotechnology Letters 8:643-648, 1986.

Preferably, the PEI coating is covalently attached to the solidsubstrate. When the solid substrate is glass or silicon, the PEI coatingmay be covalently bound to the substrate using silylating chemistry. Forexample, PEI having reactive siloxy endgroups is commercially availablefrom Gelest, Inc. (Tullytown, Pa.). Such reactive PEI may be contactedwith a glass slide or silicon wafer, and after gentle agitation, the PEIwill adhere to the substrate. Alternatively, a bifunctional silylatingreagent may be employed. According to this process, the glass or siliconsubstrate is treated with the bifunctional silylating reagent to providethe substrate with a reactive surface. PEI is then contacted with thereactive surface, and covalently binds to the surface through thebifunctional reagent.

The biomolecules being placed into the array format are originallypresent in a so-called “arraying solution”. In order to placebiomolecule in discrete regions on the PEI-coated substrate, thearraying solution preferably contains a thickening agent at aconcentration of about 35 vol % to about 80 vol % based on the totalvolume of the composition, a biomolecule which is preferably anoligonucleotide at a concentration ranging from 0.001 μg/mL to 10 μg/mL,and water.

The concentration of the thickening agent is 35% V/V to 80% V/V forliquid thickening agents such as glycerol. The preferred concentrationof thickening agent in the composition depends, to some extent, on thetemperature at which the arraying is performed. The lower the arrayingtemperature, the lower the concentration of thickening agent that needsto be used. The combination of temperature and liquid thickening agentconcentration control permits arrays to be made on most types of solidsupports (e.g., glass, wafers, nylon 6/6, nylon membranes, etc.).

The presence of a thickening agent has the additional benefit ofallowing the concurrent presence of low concentrations of various othermaterials to be present in combination with the biomolecule. For example0.001% V.V to 1% V/V of detergents may be present in the arrayingsolution. This is useful because PCR buffer contains a small amount ofTween-20 or NP-40, and it is frequently desirable to array samplenucleic acids directly from a PCR vial without prior purification of theamplicons. The use of a thickening agent permits the presence of salts(for example NaCl, KCl, or MgCl₂), buffers (for example Tris), and/orchelating reagents (for example EDTA) to also be present in the arrayingsolution. The use of a thickening agent also has the additional benefitof permitting the use of cross-linking reagents and/or organic solventsto be present in the arraying solution. As commercially obtained,cross-linking reagents are commonly dissolved in organic solvent such asDMSO, DMF, NMP, methanol, ethanol and the like. Commonly used organicsolvents can be used in arraying solutions of the invention at levels of0.05% to 20% (V/V) when thickening agents are used.

In general, the thickening agents impart increased viscosity to thearraying solution. When a proper viscosity is achieved in the arrayingsolution, the first drop is the substantially the same size as, forexample, the 100th drop deposited. When an improper viscosity is used inthe arraying solution, the first drops deposited are significantlylarger than latter drops which are deposited. The desired viscosity isbetween those of pure water and pure glycerin.

The biomolecule in the array may be a nucleic acid polymer or analogthereof, such as PNA, phosphorothioates and methylphosphonates. Nucleicacid refers to both ribonucleic acid and deoxyribonucleic acid. Thebiomolecule may comprise unnatural and/or synthetic bases. Thebiomolecule may be single or double stranded nucleic acid polymer.

A preferred biomolecule is an nucleic acid polymer, which includesoligonucleotides (up to about 100 nucleotide bases) and polynucleotides(over about 100 bases). A preferred nucleic acid polymer is formed from15 to 50 nucleotide bases. Another preferred nucleic acid polymer has 50to 1,000 nucleotide bases. The nucleic acid polymer may be a PCRproduct, PCR primer, or nucleic acid duplex, to list a few examples.However, essentially any nucleic acid type can be covalently attached toa PEI-coated surface when the nucleic acid contains a primary amine, asdisclosed below. The typical concentration of nucleic acid polymer inthe arraying solution is 0.001-10 μg/mL, preferably 0.01-1 μg/mL, andmore preferably 0.05-0.5 μg/mL.

Preferred nucleic acid polymers are “amine-modified” in that they havebeen modified to contain a primary amine at the 5′-end of the nucleicacid polymer, preferably with one or more methylene (—CH₂—) groupsdisposed between the primary amine and the nucleic acid portion of thenucleic acid polymer. Six is a preferred number of methylene groups.Amine-modified nucleic acid polymers are preferred because they can becovalently coupled to a solid support through the 5′-amine group. PCRproducts can be arrayed using 5′-hexylamine modified PCR primers.Nucleic acid duplexes can be arrayed after the introduction of amines bynick translation using aminoallyl-dUTP (Sigma, St. Louis, Mo.). Aminescan be introduced into nucleic acids by polymerases such as terminaltransferase with amino allyl-dUTP or by ligation of shortamine-containing nucleic acid polymers onto nucleic acids by ligases.

Preferably, the nucleic acid polymer is activated prior to be contactedwith the PEI coating. This can be conveniently accomplished by combiningamine-functionalized nucleic acid polymer with a multi-finctionalamine-reactive chemical such as trichlorotriazine. When the nucleic acidpolymer contains a 5′-amine group, that 5′-amine can be reacted withtrichlorotriazine, also known as cyanuric chloride (Van Ness et al.,Nucleic Acids Res. 19(2):3345-3350, 1991) Preferably, an excess ofcyanuric chloride is added to the nucleic acid polymer solution, where a10- to 1000-fold molar excess of cyanuric chloride over the number ofamines in the nucleic acid polymer in the arraying solution ispreferred. In this way, the majority of amine-terminated nucleic acidpolymers have reacted with one molecule of trichlorotriazine, so thatthe nucleic acid polymer becomes terminated with dichlorotriazine.

Preferably, the arraying solution is buffered using a common buffer suchas sodium phosphate, sodium borate, sodium carbonate, or Tris HCl. Apreferred pH range for the arraying solution is 7 to 9, with a preferredbuffer being freshly prepared sodium borate at pH 8.3 to pH 8.5. Toprepare a typical arraying solution, hexylamine-modified nucleic acidpolymer is placed in 0.2 M sodium borate, pH 8.3, at 0.1 μg/mL, to atotal volume of 50 μl. Ten μl of a 15 mg/mL solution of cyanuricchloride is then added, and the reaction is allowed to proceed for 1hour at 25 C with constant agitation. Glycerol (Gibco Brl®, GrandIsland, N.Y. is added to a final concentration of 56%.

The biomolecular arraying solutions may be applied to the PEI coating byany of the number of techniques currently used in microfabrication. Forexample, the solutions may be placed into an ink jet print head, andejected from such a head onto the coating.

A preferred approach to delivering biomolecular solution onto the PEIcoating employs a modified spring probe. Spring probes are availablefrom several vendors including Everett Charles (Pomona, Calif.),Interconnect Devices Inc. (Kansas City, Kans.) and Test ConnectionsInc., (Upland, Calif.). In order for the commercially available springprobes as described above to satisfactorily function as liquiddeposition devices according to the present invention, approximately{fraction (1/1000)}th to {fraction (5/1000)}th of an inch of metalmaterial must be removed from the tip of the probe. The process mustresult in a flat surface which is perpendicular to the longitudinal axisof the spring probe. The removal of approximately {fraction (1/1000)}thto {fraction (5/1000)}th of an inch of material from the bottom of thetip is preferred and can be accomplished easily with a very fine grainedwet stone. Specific spring probes which are commercially available andmay be modified to provide a planar tip as described above include theXP54 probe manufactured by Ostby Barton (a division of Everett Charles(Pomona, Calif.)); the SPA 25P probe manufactured by Everett Charles(Pomona, Calif.) and 43-P fluted spring probe from Test ConnectionsInc., (Upland, Calif.).

The arraying solutions as described above may be used directly in anarraying process. That is, the activated nucleic acid polymers need notbe purified away from unreacted cyanuric chloride prior to the printingstep. Typically the reaction which attaches the activated nucleic acidto the solid support is allowed to proceed for 1 to 20 hours at 20 to 50C. Preferably, the reaction time is 1 hour at 25 C.

The arrays as described herein are particularly useful in conductinghybridization assays, for example, using CMST labeled probes. However,in order to perform such assays, the amines on the solid support must becapped prior to conducting the hybridization step. This may beaccomplished by reacting the solid support with 0.1-2.0 M succinicanhydride. The preferred reaction conditions are 1.0 M succinicanhydride in 70% m-pyrol and 0.1 M sodium borate. The reaction typicallyis allowed to occur for 15 minutes to 4 hours with a preferred reactiontime of 30 minutes at 25 C. Residual succinic anhydride is removed witha 3×water wash.

The solid support is then incubated with a solution containing 0.1-5 Mglycine in 0.1-10.0 M sodium borate at pH 7-9. This step “caps” anydichloro-triazine which may be covalently bound to the PEI surface byconversion into monochlorotriazine. The preferred conditions are 0.2 Mglycine in 0.1 M sodium borate at pH 8.3. The solid support may then bewashed with detergent-containing solutions to remove unbound materials,for example, trace NMP. Preferably, the solid support is heated to 95 Cin 0.01 M NaCl, 0.05 M EDTA and 01 M Tris pH 8.0 for 5 minutes. Thisheating step removes non-covalently attached nucleic acid polymers, suchas PCR products. In the case where double strand nucleic acid arearrayed, this step also has the effect of converting the double strandto single strand form (denaturation).

The arrays are may be interrogated by probes (e.g., oligonucleotides,nucleic acid fragments, PCR products, etc.) which may be tagged with,for example CMST tags as described herein, radioisotopes, fluorophoresor biotin. The methods for biotinylating nucleic acids are well known inthe art and are adequately described by Pierce (Avidin-Biotin Chemistry:A Handbook, Pierce Chemical Company, 1992, Rockford Ill.). Probes aregenerally used at 0.1 ng/mL to 10/μg/mL in standard hybridizationsolutions that include GuSCN, GuHCl, formamide, etc. (see Van Ness andChen, Nucleic Acids Res., 19:5143-5151, 1991).

To detect the hybridization event (i.e., the presence of the biotin),the solid support is incubated with streptavidin/horseradish peroxidaseconjugate. Such enzyme conjugates are commercially available from, forexample, Vector Laboratories (Burlingham, Calif.). The streptavidinbinds with high affinity to the biotin molecule bringing the horseradishperoxidase into proximity to the hybridized probe. Unboundstreptavidin/horseradish peroxidase conjugate is washed away in a simplewashing step. The presence of horseradish peroxidase enzyme is thendetected using a precipitating substrate in the presence of peroxide andthe appropriate buffers.

A blue enzyme product deposited on a reflective surface such as a waferhas a many-fold lower level of detection (LLD) compared to that expectedfor a calorimetric substrate. Furthermore, the LLD is vastly differentfor different colored enzyme products. For example, the LLD for4-methoxynapthol (which produces a precipitated blue product) per 50 μMdiameter spot is approximately 1000 molecules, whereas a redprecipitated substrate gives an LLD about 1000-fold higher at 1,000,000molecules per 50 μM diameter spot. The LLD is determined byinterrogating the surface with a microscope (such as the Axiotechmicroscope commercially available from Zeiss) equipped with a visiblelight source and a CCD camera (Princeton Instruments, Princeton, N.J.).An image of approximately 10,000 μM×10,000 μM can be scanned at onetime.

In order to use the blue colorimetric detection scheme, the surface mustbe very clean after the enzymatic reaction and the wafer or slide mustbe scanned in a dry state. In addition, the enzymatic reaction must bestopped prior to saturation of the reference spots. For horseradishperoxidase this is approximately 2-5 minutes.

It is also possible to use chemiluminescent substrates for alkalinephosphatase or horesradish peroxidase (HRP), or fluoroescence substratesfor HRP or alkaline phosphatase. Examples include the dioxetanesubstrates for alkaline phosphatase available from Perkin Elmer orAttophos HRP substrate from JBL Scientific (San Luis Obispo, Calif.).

The following examples are offered by way of illustration, and not byway of limitation.

Unless otherwise stated, chemicals as used in the examples may beobtained from Aldrich Chemical Company, Milwaukee, Wis. The followingabbreviations, with the indicated meanings, are used herein:

ANP=3-(Fmoc-amino)-3-(2-nitrophenyl)propionic acid

NBA=4-(Fmoc-aminomethyl)-3-nitrobenzoic acid

HATU=O-7-azabenzotriazol-1-yl-NN,N′,N′-tetramethyluroniumhexafluorophosphate

DIEA=diisopropylethylamine

MCT=monochlorotriazine

NMM=4-methylmorpholine

NMP=N-methylpyrrolidone

ACT357=ACT357 peptide synthesizer from Advanced ChemTech, Inc.,Louisville, Ky.

ACT=Advanced ChemTech, Inc., Louisville, Ky.

NovaBiochem=CalBiochem-NovaBiochem International, San Diego, Calif.

TFA=Trifluoroacetic acid

Tfa=Trifluoroacetyl

iNIP=N-Methylisonipecotic acid

Tfp=Tetrafluorophenyl

DIAEA=2-(Diisopropylamino)ethylamine

MCT=monochlorotriazene

5′-AH-ODN=5′-aminohexyl-tailed oligodeoxynucleotide

EXAMPLES Example 1 PREPARATION OF ACID LABILE LINKERS FOR USE INCLEAVABLE-MW-IDENTIFIER SEQUENCING

A. Synthesis of Pentafluorophenyl Esters of Chemically Cleavable MassSpectroscopy Tags, to Liberate Tags with Carboxyl Amide Termini

FIG. 1 shows the reaction scheme.

Step A. TentaGel S AC resin (compound II; available from ACT; 1 eq.) issuspended with DMF in the collection vessel of the ACT357 peptidesynthesizer (ACT). Compound I (3 eq.), HATU (3 eq.) and DIEA (7.5 eq.)in DMF are added and the collection vessel shaken for 1 hr. The solventis removed and the resin washed with NMP (2×), MeOH (2×), and DMF (2×).The coupling of I to the resin and the wash steps are repeated, to givecompound III.

Step B. The resin (compound III) is mixed with 25% piperidine in DMF andshaken for 5 min. The resin is filtered, then mixed with 25% piperidinein DMF and shaken for 10 min. The solvent is removed, the resin washedwith NMP (2×), MeOH (2×), and DMF (2×), and used directly in step C.

Step C. The deprotected resin from step B is suspended in DMF and to itis added an FMOC-protected amino acid, containing amine finctionality inits side chain (compound IV, e.g. alpha-N-FMOC-3-(3-pyridyl)-alanine,available from Synthetech, Albany, Oreg.; 3 eq.), HATU (3 eq.), and DIEA(7.5 eq.) in DMF. The vessel is shaken for 1 hr. The solvent is removedand the resin washed with NMP (2×), MeOH (2×), and DMF (2×). Thecoupling of IV to the resin and the wash steps are repeated, to givecompound V.

Step D. The resin (compound V) is treated with piperidine as describedin step B to remove the FMOC group. The deprotected resin is thendivided equally by the ACT357 from the collection vessel into 16reaction vessels.

Step E. The 16 aliquots of deprotected resin from step D are suspendedin DMF. To each reaction vessel is added the appropriate carboxylic acidVI₁₋₁₆ (R₁₋₁₆CO₂H; 3 eq.), HATU (3 eq.), and DIEA (7.5 eq.) in DMF. Thevessels are shaken for 1 hr. The solvent is removed and the aliquots ofresin washed with NMP (2×), MeOH (2×), and DMF (2×). The coupling ofVI₁₋₁₆ to the aliquots of resin and the wash steps are repeated, to givecompounds VIII₁₋₁₆.

Step F. The aliquots of resin (compounds VII₁₋₁₆) are washed with CH₂Cl₂(3×). To each of the reaction vessels is added 1% TFA in CH₂Cl₂ and thevessels shaken for 30 min. The solvent is filtered from the reactionvessels into individual tubes. The aliquots of resin are washed withCH₂Cl₂ (2×) and MeOH (2×) and the filtrates combined into the individualtubes. The individual tubes are evaporated in vacuo, providing compoundsVIII₁₋₁₆.

Step G. Each of the free carboxylic acids VIII₁₋₁₆ is dissolved in DMF.To each solution is added pyridine (1.05 eq.), followed bypentafluorophenyl trifluoroacetate (1.1 eq.). The mixtures are stirredfor 45 min. at room temperature. The solutions are diluted with EtOAc,washed with 1 M aq. citric acid (3×) and 5% aq. NaHCO₃ (3×), dried overNa₂SO₄, filtered, and evaporated in vacuo, providing compounds IX₁₋₁₆.

B. Synthesis of Pentafluorophenyl Esters of Chemically Cleavable MassSpectroscopy Tags, to Liberate Tags with Carboxyl Acid Termini

FIG. 2 shows the reaction scheme.

Step A. 4-(Hydroxymethyl)phenoxybutyric acid (compound I; 1 eq.) iscombined with DIEA (2.1 eq.) and allyl bromide (2.1 eq.) in CHCl₃ andheated to reflux for 2 hr. The mixture is diluted with EtOAc, washedwith 1 N HCl (2×), pH 9.5 carbonate buffer (2×), and brine (1×), driedover Na₂SO₄, and evaporated in vacuo to give the allyl ester of compoundI.

Step B. The allyl ester of compound I from step A (1.75 eq.) is combinedin CH₂Cl₂ with an FMOC-protected amino acid containing aminefunctionality in its side chain (compound II, e.g.alpha-N-FMOC-3-(3-pyridyl)-alanine, available from Synthetech, Albany,Oreg.; 1 eq.), N-methylmorpholine (2.5 eq.), and HATU (1.1 eq.), andstirred at room temperature for 4 hr. The mixture is diluted withCH₂Cl₂, washed with 1 M aq. citric acid (2×), water (1×), and 5% aq.NaHCO₃ (2×), dried over Na₂SO₄, and evaporated in vacuo. Compound III isisolated by flash chromatography (CH₂Cl₂→EtOAc).

Step C. Compound III is dissolved in CH₂Cl₂, Pd(PPh₃)₄ (0.07 eq.) andN-methylaniline (2 eq.) are added, and the mixture stirred at roomtemperature for 4 hr. The mixture is diluted with CH₂Cl₂, washed with 1M aq. citric acid (2×) and water (1×), dried over Na₂SO₄, and evaporatedin vacuo. Compound IV is isolated by flash chromatography(CH₂Cl₂→EtOAc+HOAc).

Step D. TentaGel S AC resin (compound V; 1 eq.) is suspended with DMF inthe collection vessel of the ACT357 peptide synthesizer (AdvancedChemTech Inc. (ACT), Louisville, Ky.). Compound IV (3 eq.), HATU (3 eq.)and DIEA (7.5 eq.) in DMF are added and the collection vessel shaken for1 hr. The solvent is removed and the resin washed with NMP (2×), MeOH(2×), and DMF (2×). The coupling of IV to the resin and the wash stepsare repeated, to give compound VI.

Step E. The resin (compound VI) is mixed with 25% piperidine in DMF andshaken for 5 min. The resin is filtered, then mixed with 25% piperidinein DMF and shaken for 10 min. The solvent is removed and the resinwashed with NMP (2×), MeOH (2×), and DMF (2×). The deprotected resin isthen divided equally by the ACT357 from the collection vessel into 16reaction vessels.

Step F. The 16 aliquots of deprotected resin from step E are suspendedin DMF. To each reaction vessel is added the appropriate carboxylic acidVII₁₋₁₆ (R₁₋₁₆CO₂H; 3 eq.), HATU (3 eq.), and DIEA (7.5 eq.) in DMF. Thevessels are shaken for 1 hr. The solvent is removed and the aliquots ofresin washed with NMP (2×), MeOH (2×), and DMF (2×). The coupling ofVII₁₋₁₆ to the aliquots of resin and the wash steps are repeated, togive compounds VIII₁₋₁₆.

Step G. The aliquots of resin (compounds VIII₁₋₁₆) are washed withCH₂Cl₂ (3×). To each of the reaction vessels is added 1% TFA in CH₂Cl₂and the vessels shaken for 30 min. The solvent is filtered from thereaction vessels into individual tubes. The aliquots of resin are washedwith CH₂Cl₂ (2×) and MeOH (2×) and the filtrates combined into theindividual tubes. The individual tubes are evaporated in vacuo,providing compounds IX₁₋₁₆.

Step H. Each of the free carboxylic acids IX₁₋₁₆ is dissolved in DMF. Toeach solution is added pyridine (1.05 eq.), followed bypentafluorophenyl trifluoroacetate (1.1 eq.). The mixtures are stirredfor 45 min. at room temperature. The solutions are diluted with EtOAc,washed with 1 M aq. citric acid (3×) and 5% aq. NaHCO₃ (3×), dried overNa₂SO₄, filtered, and evaporated in vacuo, providing compounds X₁₋₁₆.

Example 2 DEMONSTRATION OF PHOTOLYTIC CLEAVAGE OF T—L—X

A T—L—X compound as prepared in Example 13 was irradiated with near-UVlight for 7 min at room temperature. A Rayonett fluorescence UV lamp(Southern New England Ultraviolet Co., Middletown, Conn.) with anemission peak at 350 nm is used as a source of UV light. The lamp isplaced at a 15-cm distance from the Petri dishes with samples. SDS gelelectrophoresis shows that >85% of the conjugate is cleaved under theseconditions.

Example 3 PREPARATION OF FLUORESCENT LABELED PRIMERS AND DEMONSTRATIONOF CLEAVAGE OF FLUOROPHORE

Synthesis and Purification of Oligonucleotides

The oligonucleotides (ODNs) are prepared on automated DNA synthesizersusing the standard phosphoramidite chemistry supplied by the vendor, orthe H-phosphonate chemistry (Glenn Research Sterling, Va.).Appropriately blocked dA, dG, dC, and T phosphoramnidites arecommercially available in these forms, and synthetic nucleosides mayreadily be converted to the appropriate form. The oligonucleotides areprepared using the standard phosphoramidite supplied by the vendor, orthe H-phosphonate chemistry. Oligonucleotides are purified byadaptations of standard methods. Oligonucleotides with 5′-trityl groupsare chromatographed on HPLC using a 12 micrometer, 300 # Rainin(Emeryville, Calif.) Dynamax C-8 4.2×250 mm reverse phase column using agradient of 15% to 55% MeCN in 0.1 N Et₃NH⁺OAc⁻, pH 7.0, over 20 min.When detritylation is performed, the oligonucleotides are furtherpurified by gel exclusion chromatography. Analytical checks for thequality of the oligonucleotides are conducted with a PRP-column(Alltech, Deerfield, Ill.) at alkaline pH and by PAGE.

Preparation of 2,4,6-trichlorotriazine derived oligonucleotides: 10 to1000 μg of 5′-terminal amine linked oligonucleotide are reacted with anexcess recrystallized cyanuric chloride in 10% n-methyl-pyrrolidone inalkaline (pH 8.3 to 8.5 preferably) buffer at 19° C. to 25° C. for 30 to120 minutes. The final reaction conditions consist of 0.15 M sodiumborate at pH 8.3, 2 mg/ml recrystallized cyanuric chloride and 500 ug/mlrespective oligonucleotide. The unreacted cyanuric chloride is removedby size exclusion chromatography on a G-50 Sephadex (Pharmacia,Piscataway, N.J.) column.

The activated purified oligonucleotide is then reacted with a 100-foldmolar excess of cystamine in 0.15 M sodium borate at pH 8.3 for 1 hourat room temperature. The unreacted cystamine is removed by sizeexclusion chromatography on a G-50 Sephadex column. The derived ODNs arethen reacted with amine-reactive fluorochromes. The derived ODNpreparation is divided into 3 portions and each portion is reacted witheither (a) 20-fold molar excess of Texas Red sulfonyl chloride(Molecular Probes, Eugene, Oreg.), with (b) 20-fold molar excess ofLissamine sulfonyl chloride (Molecular Probes, Eugene, Oreg.), (c)20-fold molar excess of fluorescein isothiocyanate. The final reactionconditions consist of 0.15 M sodium borate at pH 8.3 for 1 hour at roomtemperature. The unreacted fluorochromes are removed by size exclusionchromatography on a G-50 Sephadex column.

To cleave the fluorochrome from the oligonucleotide, the ODNs areadjusted to 1×10⁻⁵ molar and then dilutions are made (12, 3-folddilutions) in TE (TE is 0.01 M Tris, pH 7.0, 5 mM EDTA). To 100 μlvolumes of ODNs 25 μl of 0.01 M dithiothreitol (DTT) is added To anidentical set of controls no DDT is added. The mixture is incubated for15 minutes at room temperature. Fluorescence is measured in a blackmicrotiter plate. The solution is removed from the incubation tubes (150microliters) and placed in a black microtiter plate (DynatekLaboratories, Chantilly, Va.). The plates are then read directly using aFluoroskan II fluorometer (Flow Laboratories, McLean, Va.) using anexcitation wavelength of 495 nm and monitoring emission at 520 nm forfluorescein, using an excitation wavelength of 591 nm and monitoringemission at 612 nm for Texas Red, and using an excitation wavelength of570 nm and monitoring emission at 590 nm for lissamine.

Moles of RFU RFU RFU Fluorochrome non-cleaved cleaved free 1.0 × 10⁵M6.4 1200 1345 3.3 × 10⁶M 2.4 451 456 1.1 × 10⁶M 0.9 135 130 3.7 × 10⁷M0.3 44 48 1.2 × 10⁷M 0.12 15.3 16.0 4.1 × 10⁷M 0.14 4.9 5.1 1.4 × 10⁸M0.13 2.5 2.8 4.5 × 10⁹M 0.12 0.8 0.9

The data indicate that there is about a 200-fold increase in relativefluorescence when the fluorochrome is cleaved from the ODN.

Example 4 PREPARATION OF TAGGED M13 SEQUENCE PRIMERS AND DEMONSTRATIONOF CLEAVAGE OF TAGS

Preparation of 2,4,6-trichlorotriazine derived oligonucleotides: 1000 μgof 5′-terminal amine linked oligonucleotide(5′-hexylamine-TGTAAAACGACGGCCAGT-3″) (Seq. ID No. 1) are reacted withan excess recrystallized cyanuric chloride in 10% n-methyl-pyrrolidonealkaline (pH 8.3 to 8.5 preferably) buffer at 19 to 25- C for 30 to 120minutes. The final reaction conditions consist of 0.15 M sodium borateat pH 8.3, 2 mg/ml recrystallized cyanuric chloride and 500 ug/mlrespective oligonucleotide. The unreacted cyanuric chloride is removedby size exclusion chromatography on a G-50 Sephadex column.

The activated purified oligonucleotide is then reacted with a 100-foldmolar excess of cystamine in 0.15 M sodium borate at pH 8.3 for 1 hourat room temperature. The unreacted cystamine is removed by sizeexclusion chromatography on a G-50 Sephadex column. The derived ODNs arethen reacted with a variety of amides. The derived ODN preparation isdivided into 12 portions and each portion is reacted (25 molar excess)with the pentafluorophenyl-esters of either: (1) 4-methoxybenzoic acid,(2) 4-fluorobenzoic acid, (3) toluic acid, (4) benzoic acid, (5)indole-3-acetic acid, (6) 2,6-difluorobenzoic acid, (7) nicotinic acidN-oxide, (8) 2-nitrobenzoic acid, (9) 5-acetylsalicylic acid, (10)4-ethoxybenzoic acid, (11) cinnamic acid, (12) 3-aminonicotinic acid.The reaction is for 2 hours at 37° C. in 0.2 M NaBorate pH 8.3. Thederived ODNs are purified by gel exclusion chromatography on G-50Sephadex.

To cleave the tag from the oligonucleotide, the ODNs are adjusted to1×10⁻⁵ molar and then dilutions are made (12, 3-fold dilutions) in TE(TE is 0.01 M Tris, pH 7.0, 5 mM EDTA) with 50% EtOH (V/V). To 100 μlvolumes of ODNs 25 μl of 0.01 M dithiothreitol (DTT) is added. To anidentical set of controls no DDT is added. Incubation is for 30 minutesat room temperature. NaCl is then added to 0.1 M and 2 volumes of EtOHis added to precipitate the ODNs. The ODNs are removed from solution bycentrifugation at 14,000×G at 4° C. for 15 minutes. The supernatants arereserved, dried to completeness. The pellet is then dissolved in 25 μlMeOH. The pellet is then tested by mass spectrometry for the presence oftags.

The mass spectrometer used in this work is an external ion sourceFourier-transform mass spectrometer (FTMS). Samples prepared for MALDIanalysis are deposited on the tip of a direct probe and inserted intothe ion source. When the sample is irradiated with a laser pulse, ionsare extracted from the source and passed into a long quadrupole ionguide that focuses and transports them to an FTMS analyzer cell locatedinside the bore of a superconducting magnet.

The spectra yield the following information. Peaks varying in intensityfrom 25 to 100 relative intensity units at the following molecularweights: (1) 212.1 amu indicating 4-methoxybenzoic acid derivative, (2)200.1 indicating 4-fluorobenzoic acid derivative, (3) 196.1 amuindicating toluic acid derivative, (4) 182.1 amu indicating benzoic acidderivative, (5) 235.2 amu indicating indole-3-acetic acid derivative,(6) 218.1 amu indicating 2,6-difluorobenzoic derivative, (7) 199.1 amuindicating nicotinic acid N-oxide derivative, (8) 227.1 amu indicating2-nitrobenzamide, (9) 179.18 amu indicating 5-acetylsalicylic acidderivative, (10) 226.1 amu indicating 4-ethoxybenzoic acid derivative,(11) 209.1 amu indicating cinnamic acid derivative, (12) 198.1 amuindicating 3-aminonicotinic acid derivative.

The results indicate that the MW-identifiers are cleaved from theprimers and are detectable by mass spectrometry.

Example 5 DEMONSTRATION OF SEQUENCING USING AN HPLC SEPARATION METHOD,COLLECTING FRACTIONS, CLEAVING THE MW IDENTIFIERS, DETERMINING THE MASS(AND THUS THE IDENTITY) OF THE MW-IDENTIFIER AND THEN DEDUCING THESEQUENCE

The following oligonucleotides are prepared as described in Example 4:

DMO 767: ′5-hexylamine-TGTAAAACGACGGCCAGT-3′ (Seq. ID No. 1)

DMO 768: ′5-hexylamine-TGTAAAACGACGGCCAGTA-3′ (Seq. ID No. 2)

DMO 769: ′5-hexylamine-TGTAAAACGACGGCCAGTAT-3′ (Seq. ID No. 3)

DMO 770: ′5-hexylanine-TGTAAAACGACGGCCAGTATG-3′ (Seq. ID No. 4)

DMO 771: ′5-hexylamine-TGTAAAACGACGGCCAGTArGC-3′ (Seq. ID No. 5)

DMO 772: ′5-hexylamine-TGTAAAACGACGGCCAGTATGCA-3′ (Seq. ID No. 6)

DMO 773: ′5-hexylamine-TGTAAAACGACGGCCAGTATGCAT-3′ (Seq. ID No. 7)

DMO 774: ′5-hexylamine-TGTAAAACGACGGCCAGTATGCATG-3′ (Seq. ID No. 8)

100 μg of each of the 5′-terminal amnine-linked oligonucleotidesdescribed above are reacted with an excess recrystallized cyanuricchloride in 10% n-methyl-pyrrolidone alkaline (pH 8.3 to 8.5 preferably)buffer at 19° C. to 25° C. for 30 to 120 minutes. The final reactionconditions consist of 0.15 M sodium borate at pH 8.3, 2 mg/mlrecrystallized cyanuric chloride and 500 ug/ml respectiveoligonucleotide. The unreacted cyanuric chloride is removed by sizeexclusion chromatography on a G-50 Sephadex column.

The activated purified oligonucleotide is then reacted with a 100-foldmolar excess of cystamine in 0.15 M sodium borate at pH 8.3 for 1 hourat room temperature. The unreacted cystamine is removed by sizeexclusion chromatography on a G-50 Sephadex column. The derived ODNs arethen reacted with a particular pentafluorophenyl-ester of the following:(1) DMO 767 with 4-methoxybenzoic acid, (2) DMO 768 with 4-fluorobenzoicacid, (3) toluic acid, (4) DMO 769 with benzoic acid, (5) DMO 770 withindole-3-acetic acid, (6) DMO 771 with 2,6-difluorobenzoic acid, (7) DMO772 with nicotinic acid N-oxide, (8) DMO 773 with 2-nitrobenzoic acid.

10 ng of each of the eight derived ODNs are mixed together and then sizeseparated by HPLC. The mixture is placed in 25 μl of distilled water.The entire sample is injected on to the following column. A LiChrospher4000 DMAE, 50-10 mm column is used (EM Separations, Wakefield, R.I.).Eluent A is 20 mM Na₂HPO₄ in 20% ACN, pH7.4; Eluent B is Eluent A+1 MNaCl, pH7.4. The flowrate is for 1 ml/min and detection is UV@280 nm.The gradient is as follows: 0 min.@100% A and 0% B, 3 min.@100% A and 0%B. 15 min.@80% A and 20% B, 60 min.@0% A and 100% B, 63 min.@0% A and100% B, 65 min.@100% A and 0% B, 70 min.@100% A and 0% B. Fractions arecollected at 0.5 minute intervals.

To cleave the tags from the oligonucleotide, 100 μl of 0.05 Mdithiothreitol (DTT) is added to each fraction. Incubation is for 30minutes at room temperature. NaCl is then added to 0.1 M and 2 volumesof EtOH is added to precipitate the ODNs. The ODNs are removed fromsolution by centrifugation at 14,000×G at 4° C. for 15 minutes. Thesupematents are reserved, dried to completeness under a vacuum withcentrifugation. The pellets are then dissolved in 25 μl MeOH. The pelletis then tested by mass spectrometry for the presence of MW-identifiers.The same MALDI technique is employed as described in Example 4. Thefollowing MWs (tags) are observed in the mass spectra as a function oftime:

Fraction # Time MWs  1 0.5 none  2 1.0 none  3 1.5 none  4 2.0 none  52.5 none  6 3.0 none  7 3.5 none  8 4.0 none  9 4.5 none 10 5.0 none 115.5 none 12 6.0 none 13 6.5 none 14 7.0 none 15 7.5 none 16 8.0 none 178.5 none 18 9.0 none 19 9.5 none 20 10.0 none 21 10.5 none 22 11 none 2311.5 none 24 12 none 25 12.5 none 26 13 none 27 13.5 none 28 14 none 2914.5 none 30 15 none 31 15.5 212.1 32 16 212.1 33 16.5 212.1 34 17212.1; 200.1 35 17.5 200.1 36 18 200.1 37 18.5 200.1 38 19 200.1; 196.139 19.5 200.1; 196.1 40 20 196.1 41 20.5 196.1 42 21 196.1; 182.1 4321.5 182.1 44 22 182.1 45 22.5 182.1; 235.2 46 23 235.2 47 23.5 235.2 4824 235.2; 218.1 49 24.5 218.1 50 25 218.1 51 25.5 218.1; 199.1 52 26199.1 53 26.5 199.1; 227.1 54 27 227.1 55 27.5 227.1 56 28 none 57 28.5none 58 29 none 59 29.5 none 60 30 none

The temporal appearance of the tags is thus 212.1, 200.1, 196.1, 182.1,235.2, 218.1, 199.1, 227.1. Since 212.1 amu indicates the4-methoxybenzoic acid derivative, 200.1 indicates the 4-fluorobenzoicacid derivative, 196.1 amu indicates the toluic acid derivative, 182.1amu indicates the benzoic acid derivative, 235.2 amu indicates theindole-3-acetic acid derivative, 218.1 amu indicates the2,6-difluorobenzoic derivative, 199.1 amu indicates the nicotinic acidN-oxide derivative, 227.1 amu indicates the 2-nitrobenzamide, thesequence can be deduced as -5′-ATGCATG-3′-.

Example 6 DEMONSTRATION OF SEQUENCING OF TWO DNA SAMPLES IN A SINGLEHPLC SEPARATION METHOD

In this example, two DNA samples are sequenced in a single separationmethod.

The following oligonucleotides are prepared as described in Example 1.

DMO 767: ′5-hexylamine-TGTAAAACGACGGCCAGT-3′ (Seq. ID No. 1)

DMO 768: ′5-hexylaiine-TGTAAAACGACGGCCAGTA-3′ (Seq. ID No. 2)

DMO 769: ′5-hexylamine-TGTAAAACGACGGCCAGTAT-3′ (Seq. ID No. 3)

DMO 770: ′5-hexylamine-TGTAAAACGACGGCCAGTATG-3′ (Seq. ID No. 4)

DMO 771: ′5-hexylamine-TGTAAAACGACGGCCAGTATGC-3′ (Seq. ID No. 5)

DMO 772: ′5-hexylamine-TGTAAAACGACGGCCAGTATGCA-3′ (Seq. ID No. 6)

DMO 775: ′5-hexylamine-TGTAAAACGACGGCCAGC-3′ (Seq. ID No. 9)

DMO 776: ′5-hexylamine-TGTAAAACGACGGCCAGCG-3′ (Seq. ID No. 10)

DMO 777: ′5-hexylamine-TGTAAAACGACGGCCAGCGT-3′ (Seq. ID No. 11)

DMO 778: ′5-hexylamine-TGTAAAACGACGGCCAGCGTA-3′ (Seq. ID No. 12)

DMO 779: ′5-hexylamine-TGTAAAACGACGGCCAGCGTAC-3′ (Seq. ID No. 13)

DMO 780: ′5-hexylamine-TGTAAAACGACGGCCAGCGTACC-3′ (Seq. ID No. 14)

100 μg of each of the 5′-terminal amine-linked oligonucleotidesdescribed above are reacted with an excess recrystallized cyanuricchloride in 10% n-methyl-pyrrolidone alkaline (pH 8.3 to 8.5 preferably)buffer at 19° C. to 25° C. for 30 to 120 minutes. The final reactionconditions consist of 0.15 M sodium borate at pH 8.3, 2 mg/mlrecrystallized cyanuric chloride and 500 ug/ml respectiveoligonucleotide. The unreacted cyanuric chloride is removed by sizeexclusion chromatography on a G-50 Sephadex column.

The activated purified oligonucleotide is then reacted with a 100-foldmolar excess of cystamine in 0.15 M sodium borate at pH 8.3 for 1 hourat room temperature. The unreacted cystamine is removed by sizeexclusion chromatography on a G-50 Sephadex column. The derived ODNs arethen reacted with a particular pentafluorophenyl-ester of the following:(1) DMO 767 with 4-methoxybenzoic acid and DMO 773 with nicotinic acidN-oxide, (2) DMO 768 with 4-fluorobenzoic acid and DMO 774 with2-nitrobenzoic acid, (3) toluic acid and DMO 775 with acetylsalicylicacid, (4) DMO 769 with benzoic acid and DMO 776 with 4-ethoxybenzoicacid, (5) DMO 770 with indole-3-acetic acid and DMO 777 with cinnamicacid, (6) DMO 771 with 2,6-difluorobenzoic acid and DMO 778 with3-aminonicotinic acid. Therefore, there is one of tags for each set ofODNs.

10 ng of each of the 12 derived ODNs are mixed together and then sizeseparated by HPLC. The mixture is placed in 25 μl of distilled water.The entire sample is injected on to the following column. A LiChrospher4000 DMAE, 50-10 mm column is used (EM Separations, Wakefield, R.I.).Eluent A is 20 mM Na₂HPO₄ in 20% ACN, pH7.4; Eluent B is Eluent A+1 MNaCl, pH7.4. The flowrate is for 1 ml/min and detection is UV@280 nm.The gradient is as follows: 0 min.@100% A and 0% B, 3 min.@100% A and 0%B, 15 min.@80% A and 20% B, 60 min.@0% A and 100% B, 63 min.@0% A and100% B, 65 min.@100% A and 0% B, 70 min.@100% A and 0% B. Fractions arecollected at 0.5 minute intervals.

To cleave the tags from the oligonucleotide, 100 μl of 0.05 Mdithiothreitol (DTT) is added to each fraction. Incubation is for 30minutes at room temperature. NaCl is then added to 0.1 M and 2 volumesof EtOH is added to precipitate the ODNs. The ODNs are removed fromsolution by centrifugation at 14,000×G at 4° C. for 15 minutes. Thesupematents are reserved, dried to completeness under a vacuum withcentrifugation. The pellets are then dissolved in 25 μl MeOH. The pelletis then tested by mass spectrometry for the presence of tags. The sameMALDI technique is employed as described in Example 4. The following MWs(tags) are observed in the mass spectra as a function of time:

Fraction # Time MWs Fraction # Time MWs 1 0.5 none 31 15.5 212.1, 199.12 1.0 none 32 16 212.1, 199.1 3 1.5 none 33 16.5 212.1, 199.1 4 2.0 none34 17 212.1; 200.1, 199.1, 227.1 5 2.5 none 35 17.5 200.1, 199.1, 227.16 3.0 none 36 18 200.1, 227.1 7 3.5 none 37 18.5 200.1, 227.1, 179.18 84.0 none 38 19 200.1; 196.1, 179.18 9 4.5 none 39 19.5 200.1; 196.1,179.18 10 5.0 none 40 20 196.1, 179.18, 226.1 11 5.5 none 41 20.5 196.1,226.1 12 6.0 none 42 21 196.1; 182.1, 226.1 13 6.5 none 43 21.5 182.1,226.1, 209.1 14 7.0 none 44 22 182.1, 209.1 15 7.5 none 45 22.5 182.1;235.2, 209.1, 198.1 16 8.0 none 46 23 235.2, 198.1 17 8.5 none 47 23.5235.2, 198.1 18 9.0 none 48 24 235.2;, 198.1, 218.1 19 9.5 none 49 24.5218.1 20 10.0 none 50 25 218.1 21 10.5 none 51 25.5 none 22 11 none 5226 none 23 11.5 none 53 26.5 none 24 12 none 54 27 none 25 12.5 none 5527.5 none 26 13 none 56 28 none 27 13.5 none 57 28.5 none 28 14 none 5829 none 29 14.5 none 59 29.5 none 30 15 none 60 30 none

The temporal appearance of the tags for set #1 is 212.1, 200.1, 196.1,182.1, 235.2, 218.1, 199.1, 227.1, and the temporal appearance of tagsfor set #2 is 199.1, 227.1, 179.1, 226.1, 209.1, 198.1. Since 212.1 amuindicates the 4-methoxybenzoic acid derivative, 200.1 indicates the4-fluorobenzoic acid derivative, 196.1 amu indicates the toluic acidderivative, 182.1 amu indicates the benzoic acid derivative, 235.2 amuindicates the indole-3-acetic acid derivative, 218.1 amu indicates the2,6-difluorobenzoic derivative, 199.1 amu indicates the nicotinic acidN-oxide derivative, 227.1 amu indicates the 2-nitrobenzamide, 179.18 amuindicates the 5-acetylsalicylic acid derivative, 226.1 amu indicates the4-ethoxybenzoic acid derivative, 209.1 amu indicates the cinnamic acidderivative, and 198.1 amu indicates the 3-aminonicotinic acid, the firstsequence can be deduced as -5′-TATGCA-3′- and the second sequence can bededuced as -5′-CGTACC-3′-. Thus, it is possible to sequence more thanone DNA sample per separation step.

Example 7 PREPARATION OF A SET OF COMPOUNDS OF THE FORMULAR₁₋₃₆-LYS(ε-INIP)-ANP-TFP

FIG. 3 illustrates the parallel synthesis of a set of 36 T—L—X compounds(X=L_(h)), where L_(h) is an activated ester (specifically,tetrafluorophenyl ester), L² is an ortho-nitrobenzylamine group with L³being a methylene group that links L_(h) and L², T has a modularstructure wherein the carboxylic acid group of lysine has been joined tothe nitrogen atom of the L² benzylamine group to form an amide bond, anda variable weight component R₁₋₃₆, (where these R groups correspond toT² as defined herein, and may be introduced via any of the specificcarboxylic acids listed herein) is bonded through the α-amino group ofthe lysine, while a mass spec sensitivity enhancer group (introduced viaN-methylisonipecotic acid) is bonded through the C-amino group of thelysine.

Referring to FIG. 3:

Step A. NovaSyn HMP Resin (available from NovaBiochem; 1 eq.) issuspended with DMF in the collection vessel of the ACT357. Compound I(ANP available from ACT; 3 eq.), HATU (3 eq.) and NMM (7.5 eq.) in DMFare added and the collection vessel shaken for 1 hr. The solvent isremoved and the resin washed with NMP (2×), MeOH (2×), and DMF (2×). Thecoupling of I to the resin and the wash steps are repeated, to givecompound II.

Step) B. The resin (compound II) is mixed with 25% piperidine in DMF andshaken for 5 min. The resin is filtered, then mixed with 25% piperidinein DMF and shaken for 10 min. The solvent is removed, the resin washedwith NMP (2×), MeOH (2×), and DMF (2×), and used directly in step C.

Step C. The deprotected resin from step B is suspended in DMF and to itis added an FMOC-protected amino acid, containing a protected aminefunctionality in its side chain (Fmoc-Lysine(Aloc)-OH, available fromPerSeptive Biosystems; 3 eq.), HATU (3 eq.), and NMM (7.5 eq.) in DMF.The vessel is shaken for 1 hr. The solvent is removed and the resinwashed with NMP (2×), MeOH (2×), and DMF (2×). The coupling ofFmoc-Lys(Aloc)-OH to the resin and the wash steps are repeated, to givecompound IV.

Step D. The resin (compound IV) is washed with CH₂Cl₂ (2×), and thensuspended in a solution of (PPh₃)₄Pd (0) (0.3 eq.) and PhSiH₃ (10 eq.)in CH₂Cl₂. The mixture is shaken for 1 hr. The solvent is removed andthe resin is washed with CH₂Cl₂ (2×). The palladium step is repeated.The solvent is removed and the resin is washed with CH₂Cl₂ (2×),N,N-diisopropylethylammonium diethyldithiocarbamate in DMF (2×), DMF(2×) to give compound V.

Step E. The deprotected resin from step D is coupled withN-methylisonipecotic acid as described in step C to give compound VI.

Step) F. The Fmoc protected resin VI is divided equally by the ACT357from the collection vessel into 36 reaction vessels to give compoundsVI₁₋₃₆.

Step G. The resin (compounds VIl₁₋₃₆) is treated with piperidine asdescribed in step B to remove the FMOC group.

Step H. The 36 aliquots of deprotected resin from step G are suspendedin DMF. To each reaction vessel is added the appropriate carboxylic acid(R₁₋₃₆CO₂H; 3 eq.), HATU (3 eq.), and NMM (7.5 eq.) in DMF. The vesselsare shaken for 1 hr. The solvent is removed and the aliquots of resinwashed with NMP (2×), MeOH (2×), and DMF (2×). The coupling of R₁₋₃₆CO₂Hto the aliquots of resin and the wash steps are repeated, to givecompounds VIII₁₋₃₆.

Step I. The aliquots of resin (compounds VIII₁₋₃₆) are washed withCH₂Cl₂ (3×). To each of the reaction vessels is added 90:5:5TFA:H20:CH₂Cl₂ and the vessels shaken for 120 min. The solvent isfiltered from the reaction vessels into individual tubes. The aliquotsof resin are washed with CH₂Cl₂ (2×) and MeOH (2×) and the filtratescombined into the individual tubes. The individual tubes are evaporatedin vacuo, providing compounds IX₁₋₃₆.

Step J. Each of the free carboxylic acids IX₁₋₃₆ is dissolved in DMF. Toeach solution is added pyridine (1.05 eq.), followed bytetrafluorophenyl trifluoroacetate (1.1 eq.). The mixtures are stirredfor 45 min. at room temperature. The solutions are diluted with EtOAc,washed with 5% aq. NaHCO₃ (3×), dried over Na₂SO₄, filtered, andevaporated in vacuo, providing compounds X₁₋₃₆.

Example 8 PREPARATION OF A SET OF COMPOUNDS OF THE FORMULAR₁₋₃₆-LYS(ε-INIP)-NBA-TFP

FIG. 4 illustrates the parallel synthesis of a set of 36 T—L—X compounds(X=L_(h)), where L_(h) is an activated ester (specifically,tetrafluorophenyl ester), L² is an ortho-nitrobenzylamine group with L³being a direct bond between L_(h) and L², where L_(h) is joined directlyto the aromatic ring of the L² group, T has a modular structure whereinthe carboxylic acid group of lysine has been joined to the nitrogen atomof the L² benzylamine group to form an amide bond, and a variable weightcomponent R₁₋₃₆, (where these R groups correspond to T² as definedherein, and may be introduced via any of the specific carboxylic acidslisted herein) is bonded through the ax-amino group of the lysine, whilea mass spec enhancer group (introduced via N-methylisonipecotic acid) isbonded through the E-amino group of the lysine.

Referring to FIG. 4:

Step A. NovaSyn HMP Resin is coupled with compound I (NBA preparedaccording to the procedure of Brown et al., Molecular Diversity, 1, 4(1995)) according to the procedure described in step A of Example 7, togive compound II.

Steps B-J. The resin (compound II) is treated as described in steps B-Jof Example 7 to give compounds X₁₋₃₆.

Example 9 PREPARATION OF A SET OF COMPOUNDS OF THE FORMULA I NIP-L YS(ε-R₁₋₃₆)-ANP-TFP

FIG. 5 illustrates the parallel synthesis of a set of 36 T—L—X compounds(X=L_(h)), where L_(h) is an activated ester (specifically,tetrafluorophenyl ester), L² is an ortho-nitrobenzylamine group with L³being a methylene group that links L_(h) and L², T has a modularstructure wherein the carboxylic acid group of lysine has been joined tothe nitrogen atom of the L² benzylamine group to form an amide bond, anda variable weight component R₁₋₃₆, (where these R groups correspond toT² as defined herein, and may be introduced via any of the specificcarboxylic acids listed herein) is bonded through the ε-amino group ofthe lysine, while a mass spec sensitivity enhancer group (introduced viaN-methylisonipecotic acid) is bonded through the α-amino group of thelysine.

Referring to FIG. 5:

Steps A-C. Same as in Example 7.

Step D. The resin (compound IV) is treated with piperidine as describedin step B of Example 7 to remove the FMOC group.

Step E. The deprotected α-amine on the resin in step D is coupled withN-30 methylisonipecotic acid as described in step C of Example 7 to givecompound V.

Step F. Same as in Example 7.

Step G. The resin (compounds VI₁₋₃₆) are treated with palladium asdescribed in step D of Example 7 to remove the Aloc group.

Steps H-J. The compounds X₁₋₃₆ are prepared in the same manner as inExample 7.

Example 10 PREPARATION OF A SET OF COMPOUNDS OF THE FORMULAR₁₋₃₆-GLU(γ-DIAEA)-ANP-TFP

FIG. 6 illustrates the parallel synthesis of a set of 36 T—L—X compounds(X=L_(h)), where L_(h) is an activated ester (specifically,tetrafluorophenyl ester), L² is an ortho-nitrobenzylamine group with L³being a methylene group that links L_(h) and L², T has a modularstructure wherein the α-carboxylic acid group of glutamatic acid hasbeen joined to the nitrogen atom of the L² benzylamine group to form anamide bond, and a variable weight component R₁₋₃₆, (where these R groupscorrespond to T² as defined herein, and may be introduced via any of thespecific carboxylic acids listed herein) is bonded through the aα-aminogroup of the glutamic acid, while a mass spec sensitivity enhancer group(introduced via 2-(diisopropylamino)ethylamine) is bonded through theγ-carboxylic acid of the glutamic acid.

Referring to FIG. 6:

Steps A-B. Same as in Example 7.

Step C. The deprotected resin (compound III) is coupled toFmoc-Glu-(OAI)-OH using the coupling method described in step C ofExample 7 to give compound IV.

Step D. The allyl ester on the resin (compound IV) is washed with CH₂Cl₂(2×) and mixed with a solution of (PPh₃)₄Pd (0) (0.3 eq.) andN-methylaniline (3 eq.) in CH₂Cl₂. The mixture is shaken for 1 hr. Thesolvent is removed and the resin is washed with CH₂Cl₂ (2×). Thepalladium step is repeated. The solvent is removed and the resin iswashed with CH₂Cl₂ (2×), N,N-diisopropylethylammoniumdiethyldithiocarbamate in DMF (2×), DMF (2×) to give compound V.

Step E. The deprotected resin from step D is suspended in DMF andactivated by mixing HATU (3 eq.), and NMM (7.5 eq.). The vessels areshaken for 15 minutes. The solvent is removed and the resin washed withNMP (1×). The resin is mixed with 2-(diisopropylamino)ethylamine (3 eq.)and NMM (7.5 eq.). The vessels are shaken for 1 hour. The coupling of2-(diisopropylamino)ethylamine to the resin and the wash steps arerepeated, to give compound VI.

Steps F-J. Same as in Example 7.

Example 11 PREPARATION OF A SET OF COMPOUNDS OF THE FORMULAR₁₋₃₆-LYS(ε-INIP)-ANP-LYS(ε-NH₂)—NH₂

FIG. 7 illustrates the parallel synthesis of a set of 36 T—L—X compounds(X=L_(h)), where L_(h) is an amine (specifically, the ε-amino group of alysine-derived moiety), L² is an ortho-nitrobenzylamine group with L³being a carboxamido-substituted alkyleneaminoacylalkylene group thatlinks L_(h) and L², T has a modular structure wherein the carboxylicacid group of lysine has been joined to the nitrogen atom of the L²benzylamine group to form an amide bond, and a variable weight componentR₁₋₃₆, (where these R groups correspond to T² as defined herein, and maybe introduced via any of the specific carboxylic acids listed herein) isbonded through the α-amino group of the lysine, while a mass specsensitivity enhancer group (introduced via N-methylisonipecotic acid) isbonded through the ε-amino group of the lysine.

Referring to FIG. 7:

Step A. Fmoc-Lys(Boc)-SRAM Resin (available from ACT; compound I) ismixed with 25% piperidine in DMF and shaken for 5 min. The resin isfiltered, then mixed with 25% piperidine in DMF and shaken for 10 min.The solvent is removed, the resin washed with NMP (2×), MeOH (2×), andDMF (2×), and used directly in step B.

Step B. The resin (compound II), ANP (available from ACT; 3 eq.), HATU(3 eq.) and NMM (7.5 eq.) in DMF are added and the collection vesselshaken for 1 hr. The solvent is removed and the resin washed with NMP(2×), MeOH (2×), and DMF (2×). The coupling of I to the resin and thewash steps are repeated, to give compound III.

Steps C-J. The resin (compound III) is treated as in steps B-I inExample 7 to give compounds X₁₋₃₆.

Example 12 PREPARATION OF A SET OF COMPOUNDS OF THE FORMULAR₁₋₃₆-LYS(ε-TFA)-L YS(ε-IINP)-ANP-TFP

FIG. 8 illustrates the parallel synthesis of a set of 36 T—L—X compounds(X=L_(h)), where L_(h) is an activated ester (specifically,tetrafluorophenyl ester), L² is an ortho-nitrobenzylamine group with L³being a methylene group that links L_(h) and L², T has a modularstructure wherein the carboxylic acid group of a first lysine has beenjoined to the nitrogen atom of the L² benzylamine group to form an amidebond, a mass spec sensitivity enhancer group (introduced viaN-methylisonipecotic acid) is bonded through the ε-amino group of thefirst lysine, a second lysine molecule has been joined to the firstlysine through the α-amino group of the first lysine, a molecular weightadjuster group (having a trifluoroacetyl structure) is bonded throughthe ε-amino group of the second lysine, and a variable weight componentR₁₋₃₆, (where these R groups correspond to T² as defined herein, and maybe introduced via any of the specific carboxylic acids listed herein) isbonded through the α-amino group of the second lysine.

Referring to FIG. 8:

Steps A-E. These steps are identical to steps A-E in Example 7.

Step F. The resin (compound VI) is treated with piperidine as describedin step B in Example 7 to remove the FMOC group.

Step G. The deprotected resin (compound VII) is coupled toFmoc-Lys(Tfa)—OH using the coupling method described in step C ofExample 7 to give compound VIII.

Steps H-K. The resin (compound VIII) is treated as in steps F-J inExample 7 to give compounds XI₁₋₃₆.

Example 13 PREPARATION OF A SET OF COMPOUNDS OF THE FORMULAR₁₋₃₆-LYS(ε-INIP)-ANP-5′-AH-ODN

FIG. 9 illustrates the parallel synthesis of a set of 36 T—L—X compounds(X=MOI, where MOI is a nucleic acid fragment, ODN) derived from theesters of Example 7 (the same procedure could be used with other T—L—Xcompounds wherein X is an activated ester). The MOI is conjugated to T—Lthrough the 5′ end of the MOI, via a phosphodiester—alkyleneamine group.

Referring to FIG. 9:

Step A. Compounds XII₁₋₃₆ are prepared according to a modifiedbiotinylation procedure in Van Ness et al., Nucleic Acids Res., 19, 3345(1991). To a solution of one of the 5′-aminohexyl oligonucleotides(compounds XI₁₋₃₆, 1 mg) in 200 mM sodium borate (pH 8.3, 250 mL) isadded one of the Tetrafluorophenyl esters (compounds X₁₋₃₆ from ExampleA, 100-fold molar excess in 250 mL of NMP). The reaction is incubatedovernight at ambient temperature. The unreacted and hydrolyzedtetrafluorophenyl esters are removed from the compounds XII₁₋₃₆ bySephadex G-50 chromatography.

Example 14 PREPARATION OF A SET OF COMPOUNDS OF THE FORMULAR₁₋₃₆-LYS(ε-INIP)-ANP-LYS(ε-(MCT-5′-AH-ODN))—NH₂

FIG. 10 illustrates the parallel synthesis of a set of 36 T—L—Xcompounds (X=MOI, where MOI is a nucleic acid fragment, ODN) derivedfrom the amines of Example 11 (the same procedure could be used withother T—L—X compounds wherein X is an amine). The MOI is conjugated toT—L through the 5′ end of the MOI, via a phosphodiester—alkyleneaminegroup.

Referring to FIG. 10:

Step A. The5′-[6-(4,6-dichloro-1,3,5-triazin-2-ylamino)hexyl]oligonucleotidesXII₁₋₃₆ are prepared as described in Van Ness et al., Nucleic AcidsRes., 19, 3345 (1991).

Step B. To a solution of one of the5′-[6-(4,6-dichloro-1,3,5-triazin-2-ylamino)hexyl]oligonucleotides(compounds XII₁₋₃₆) at a concentration of 1 mg/ml in 100 mM sodiumborate (pH 8.3) was added a 100-fold molar excess of a primary amineselected from R₁₋₃₆-Lys(e-iNIP)-ANP-Lys(e-NH₂)—NH₂ (compounds X₁₋₃₆ fromExample 11). The solution is mixed overnight at ambient temperature. Theunreacted amine is removed by ultrafiltration through a 3000 MW cutoffmembrane (Amicon, Beverly, Mass.) using H₂O as the wash solution (3×).The compounds XIII₁₋₃₆ are isolated by reduction of the volume to 100mL.

Example 15 DEMONSTRATION OF SEQUENCING USING A CE SEPARATION METHOD,COLLECTING FRACTIONS, CLEAVING THE TAG, DETERMINING THE MASS (AND THUSTHE IDENTITY) OF THE TAG AND THEN DEDUCING THE SEQUENCE

In this example, two DNA samples are sequenced in a single separationmethod.

CE Instrumentation

The CE instrument is a breadboard version of the instrument availablecommercially from Applied Biosystems, Inc. (Foster City, Calif.). Itconsists of Plexiglas boxes enclosing two buffer chambers, which can bemaintained at constant temperature with a heat control unit. The voltagenecessary for electrophoresis is provided by a high-voltage power supply(Gamma High Voltage Research, Ormond Beach, Fla.) with a magnetic safetyinterlock, and a control unit to vary the applied potential. Sampleinjections for open tube capillaries are performed by use of a handvacuum pump to generate a pressure differential across the capillary(vacuum injection). For gel-filled capillaries, samples areelectrophoresed into the tube by application of an electric field(electrokinetic injection).

Preparation of Gel-Filled Capillaries

Fifty-centimeter fused silica capillaries (375 mm o.d., 50 mm i.d.,Polymicro Technologies, Phoenix, Ariz.) with detector windows (where thepolyimide coating has been removed from the capillary) at 25 cm are usedin the separations. The inner surface of the capillaries are derivatizedwith (methyacryloxypropyl)trimethoxysilane (MAPS) (Sigma, St. Louis,Mo.) to permit covalent attachment of the gel to the capillary wall(Nashabeh et al., Anal Chem63:2148, 1994). Briefly, the capillaries arecleaned by successively flowing trifluoracetic acid, deionized water,and acetone through the column. After the acetone wash, 0.2% solution ofMAPS in 50/50 water/ethanol solution is passed through the capillary andleft at room temperature for 30 min. The solution is removed byaspiration and the tubes are dried for 30 min under an infrared heatlamp.

Gel-filled capillaries are prepared under high pressure by amodification of the procedure described by Huang et al. (J.Chromatography 600:289, 1992). Four percent poly(acrylamide) gels with5% cross linker and 8.3 urea are used for all the studies reported here.A stock solution is made by dissolving 3.8 g of acrylamide, 0.20 g ofN,N′-methylenebis (acrylamide), and 50 g of urea into 100 mL of TBEbuffer (90 mM Tris borate, pH 8.3, 0.2 mM EDTA). Cross linking isinitiated with 10 mL of N,N,N′,N′-tetramethylethylenediamine (TEMED) and250 mL of 10% ammonium persulfate solution. The polymerizing solution isquickly passed into the derivatized column. Filled capillaries are thenplaced in a steel tube 1×m×⅛ in. i.d.×¼ in. o.d. filled with water, andthe pressure is raised to 400 bar by using a HPLC pump and maintained atthat pressure overnight. The pressure is gradually released and thecapillaries are removed. A short section of capillary from each end ofthe column is removed before use.

Separation and Detection of DNA Fragments

Analysis of DNA sequencing reactions separated by conventionalelectrophoresis is performed on an ABI 370A DNA sequencer. Thisinstrument uses a slab denaturing urea poly(acrylamide) gel 0.4 mm thickwith a distance of 26 cm from the sample well to the detection region,prepared according to the manufacturers instructions. DNA sequencingreactions are prepared as described by the manufacturer utilizing Taqpolymerase (Promega Corp., Madison, Wis.) and are performed on M13mp19single-stranded DNA template prepared by standard procedures. Sequencingreactions are stored at −20° C. in the dark and heated at 90° C. for 3min in formamide just prior to sample loading. They are loaded on the370A with a pipetman according to the manufacturers instructions and onthe CE by electrokinetic injection at 10,000 V for 10 seconds. Ten μlfractions are collected during the run by removing the all the liquid atthe bottom electrode and replacing it with new electolyte.

To cleave the tag from the oligonucleotide, 100 μl of 0.05 Mdithiothreitol (DTT) is added to each fraction. Incubation is for 30minutes at room temperature. NaCl is then added to 0.1 M and 2 volumesof EtOH is added to precipitate the ODNs. The ODNs are removed fromsolution by centrifugation at 14,000×G at 4° C. for 15 minutes. Thesupernatents are reserved, dried to completeness under a vacuum withcentrifugation. The pellets are then dissolved in 25 μl MeOH. The pelletis then tested by mass spectrometry for the presence of tags. The sameMALDI technique is employed as described in Example 4. The following MWs(tags) are observed in the mass spectra as a function of time:

Fraction # Time MWs Fraction # Time MWs 1 1 none 31 31 212.1, 2 2 none32 32 212.1, 199.1 3 3 none 33 33 212.1, 199.1 4 4 none 34 34 212.1;200.1, 199.1, 227.1 5 5 none 35 35 200.1, 199.1, 227.1 6 6 none 36 36200.1, 227.1 7 7 none 37 37 200.1, 227.1, 179.18 8 8 none 38 38 200.1;196.1, 179.18 9 9 none 39 39 200.1; 196.1, 179.18 10 10 none 40 40196.1, 179.18, 226.1 11 11 none 41 41 196.1, 226.1 12 12 none 42 42196.1; 182.1, 226.1 13 13 none 43 43 182.1, 226.1, 209.1 14 14 none 4444 182.1, 209.1 15 15 none 45 45 182.1; 235.2, 209.1, 198.1 16 16 none46 46 235.2, 198.1 17 17 none 47 47 235.2, 198.1 18 18 none 48 48235.2;, 198.1, 218.1 19 19 none 49 49 218.1 20 20 none 50 50 218.1 21 21none 51 51 none 22 22 none 52 52 none 23 23 none 53 53 none 24 24 none54 54 none 25 25 none 55 55 none 26 26 none 56 56 none 27 27 none 57 57none 28 28 none 58 58 none 29 29 212.1 59 59 none 30 30 212.1 60 60 none

The temporal appearance of the tags for set #1 is 212.1, 200.1, 196.1,182.1, 235.2, 218.1, 199.1, 227.1, and the temporal appearance of tagsfor set #2 is 199.1, 227.1, 179.1, 226.1, 209.1, 198.1. Since 212.1 amuindicates the 4-methoxybenzoic acid derivative, 200.1 indicates the4-fluorobenzoic acid derivative, 196.1 amu indicates the toluic acidderivative, 182.1 amu indicates the benzoic acid derivative, 235.2 amuindicates the indole-3-acetic acid derivative, 218.1 amu indicates the2,6-difluorobenzoic derivative, 199.1 amu indicates the nicotinic acidN-oxide derivative, 227.1 arnu indicates the 2-nitrobenzamide, 179.18amu indicates the 5-acetylsalicylic acid derivative, 226.1 amu indicatesthe 4-ethoxybenzoic acid derivative, 209.1 amu indicates the cinnamicacid derivative, and 198.1 amu indicates the 3-aminonicotinic acid, thefirst sequence can be deduced as -5′-TATGCA-3′- and the second sequencecan be deduced as -5′-CGTACC-3′-. Thus, it is possible to sequence morethan one DNA sample per separation step.

Example 16 DEMONSTRATION OF THE SIMULTANEOUS DETECTION OF MULTIPLE TAGSBY MASS SPECTROMETRY

This example provides a description of the ability to simultaneouslydetect multiple compounds (tags) by mass spectrometry. In thisparticular example, 31 compounds are mixed with a matrix, deposited anddried on to a solid support and then desorbed with a laser. Theresultant ions are then introduced in a mass spectrometer.

The following compounds (purchased from Aldrich, Milwaukee, Wis.) aremixed together on an equal molar basis to a final concentration of 0.002M (on a per compound) basis: benzamide (121.14), nicotinamide (122.13),pyrazinamide (123.12), 3-amino-4-pyrazolecarboxylic acid (127.10),2-thiophenecarboxamide (127.17), 4-aminobenzamide (135.15), tolumide(135.17), 6-methylnicotinamide (136.15), 3-aminonicotinamide (137.14),nicotinamide N-oxide (138.12), 3-hydropicolinamide (138.13),4-fluorobenzamide (139.13), cinnamamide (147.18), 4-methoxybenzamide(151.17), 2,6-difluorbenzamide (157.12),4-amino-5-imidazole-carboxyamide (162.58), 3,4-pyridine-dicarboxyamide(165.16), 4-ethoxybenzamide (165.19), 2,3-pyrazinedicarboxamide(166.14), 2-nitrobenzamide (166.14), 3-fluoro-4-methoxybenzoic acid(170.4), indole-3-acetamide (174.2), 5-acetylsalicylamide (179.18),3,5-dimethoxybenzamide (181.19), 1-naphthaleneacetamide (185.23),8-chloro-3,5-diamino-2-pyrazinecarboxyamide (187.59),4-trifluoromethyl-benzamide (189.00),5-amino-5-phenyl-4-pyrazole-carboxamide (202.22),1-methyl-2-benzyl-malonamate (207.33),4-amino-2,3,5,6-tetrafluorobenzamide (208.11), 2,3-napthlenedicarboxylicacid (212.22). The compounds are placed in DMSO at the concentrationdescribed above. One μl of the material is then mixed withalpha-cyano-4-hydroxy cinnamic acid matrix (after a 1:10,000 dilution)and deposited on to a solid stainless steel support.

The material is then desorbed by a laser using the Protein TOF MassSpectrometer (Bruker, Manning Park, Mass.) and the resulting ions aremeasured in both the linear and reflectron modes of operation. Thefollowing m/z values are observed (FIG. 11):

121.1→benzamide (121.14)

122.1→nicotinamide (122.13)

123.1→pyrazinamide (123.12)

124.1

125.2

127.3→3-amino-4-pyrazolecarboxylic acid (127.10)

127.2→2-thiophenecarboxamide (127.17)

135.1→4-aminobenzamide (135.15)

135.1→tolumide (135.17)

136.2→6-methylnicotinamide (136.15)

137.1→3-aminonicotinamide (137.14)

138,2→nicotinamide N-oxide (138.12)

138.2→3-hydropicolinamide (138.13)

139.2→4-fluorobenzamide (139.13)

140.2

147.3→cinnamamide (147.18)

148.2

149.2 4-methoxybenzamide (151.17)

152.2 2,6-difluorbenzamide (157.12)

158.3 4-amino-5-imidazole-carboxyamide (162.58)

163.3

165.2→3,4-pyridine-dicarboxyamide (165.16)

165.2→4-ethoxybenzamide (165.19)

166.2→2,3-pyrazinedicarboxamide (166.14)

166.2→2-nitrobenzamide (166.14) 3-fluoro-4-methoxybenzoic acid (170.4)

171.1

172.2

173.4 indole-3-acetamide (174.2)

178.3

179.3→5-acetylsalicylamide (179.18)

181.2→3,5-dimethoxybenzamide (181.19)

182.2→1-naphthaleneacetamide (185.23)

186.2 8-chloro-3,5-diamino-2-pyrazinecarboxyamide (187.59)

188.2

189.2 →4-trifluoromethyl-benzamide (189.00)

190.2

191.2

192.3 5-amino-5-phenyl-4-pyrazole-carboxamide (202.22)

203.2

203.4 1-methyl-2-benzyl-malonamate (207.33)4-amino-2,3,5,6-tetrafluorobenzamide (208.11)

212.2→2,3-napthlenedicarboxylic acid (212.22).

219.3

221.2

228.2

234.2

237.4

241.4

The data indicate that 22 of 31 compounds appeared in the spectrum withthe anticipated mass, 9 of 31 compounds appeared in the spectrum with an+H mass (1 atomic mass unit, amu) over the anticipated mass. The latterphenomenon is probably due to the protonation of an amine within thecompounds. Therefore 31 of 31 compounds are detected by MALDI MassSpectroscopy. More importantly, the example demonstrates that multipletags can be detected simultaneously by a spectroscopic method.

The alpha-cyano matrix alone (FIG. 12) gave peaks at 146.2, 164.1,172.1, 173.1, 189.1, 190.1, 191.1, 192.1, 212.1, 224.1, 228.0, 234.3.Other identified masses in the spectrum are due to contaminants in thepurchased compounds as no effort was made to further purify thecompounds.

Example 17 A PROCEDURE FOR SEQUENCING WITH MW-IDENTIFIER-LABELEDPRIMERS, RADIOLABELED PRIMERS,MW-IDENTIFIER-LABELED-DIDEOXY-TERMINATORS, FLUORESCENT-PRIMERS ANDFLUORESCENT-DIDEOXY-TERMINATORS

A. Preparation Sequencing Gels and Electrophoresis

The protocol is as follows. Prepare 8 M urea, polyacrylamide gelsaccording to the following recipes (100 ml) for 4%, 6%, or 8%polyacrylamide.

4% 5% 6% urea 48 g 48 48 g 40% acrylamide/bisacrylamide 10 ml 12.5 ml 15ml 10X MTBE 10 ml 10 ml 10 ml ddH₂O 42 ml 39.5 ml 37 ml 15% APS 500 μl500 μl 500 μl TEMED 50 μl 50 μl 50 μl

Urea (5505UA) is obtained from Gibco/BRL (Gaithersburg, Md.). All othermaterials are obtained from Fisher (Fair Lawn, N.J.). Briefly, urea,MTBE buffer and water are combined, incubated for 5 minutes at 55° C.,and stirred to dissolve the urea. The mixture is cooled briefly,acrylamide/bis-acrylamide solution is added and mixed, and the entiremixture is degassed under vacuum for 5 minutes. APS and TEMEDpolymerization agents are added with stirring. The complete gel mix isimmediately poured in between the taped glass plates with 0.15 mmspacers. Plates are prepared by first cleaning with ALCONOX™ (New York,N.Y. detergent and hot water, are rinsed with double distilled water,and dried. Typically, the notched glass plate is treated with asilanizing reagent and then rinsed with double distilled water. Afterpouring, the gel is immediately laid horizontally, the well forming combis inserted, clamped into place, and the gel allowed to polymerize forat least 30 minutes. Prior to loading, the tape around the bottom of thegel and the well-forning comb is removed. A vertical electrophoresisapparatus is then assembled by clamping the upper and lower bufferchambers to the gel plates, and adding 1× MTBE electrophoresis buffer tothe chambers. Sample wells are flushed with a syringe containing runningbuffer, and immediately prior to loading each sample, the well isflushed with running buffer using gel loading tips to remove urea. Oneto two microliters of sample is loaded into each well using a Pipetteman(Rainin, Emeryville, Calif.) with gel-loading tips, and thenelectrophoresed according the following guidelines (duringelectrophoresis, the gel is cooled with a fan):

termination electrophoresis reaction polyacrylamide gel conditions short5%, 0.15 mm × 50 cm × 20 cm 2.25 hours at 22 mA long 4%, 0.15 mm × 70 cm× 20 cm 8-9 hours at 15 mA long 4%, 0.15 mm × 70 cm × 20 cm 20-24 hoursat 15 mA

Each base-specific sequencing reaction terminated (with the shorttermination) mix is loaded onto a 0.15 mm×50 cm×20 cm denaturing 5%polyacrylamide gel; reactions terminated with the long termination mixtypically are divided in half and loaded onto two 0.15 mm×70 cm×20 cmdenaturing 4% polyacrylamide gels.

After electrophoresis, buffer is removed from the wells, the tape isremoved, and the gel plates separated. The gel is transferred to a 40cm×20 cm sheet of 3MM Whatman paper, covered with plastic wrap, anddried on a Hoefer (San Francisco, Calif.) gel dryer for 25 minutes at80° C. The dried gel is exposed to Kodak (New Haven, Conn.) XRP-1 film.Depending on the intensity of the signal and whether the radiolabel is³²P or ³⁵S, exposure times vary from 4 hours to several days. Afterexposure, films are developed by processing in developer and fixersolutions, rinsed with water, and air dried. The autoradiogram is thenplaced on a light-box, the sequence is manually read, and the data typedinto a computer.

Taq-polymerase catalyzed cycle sequencing using labeled primers. Eachbase-specific cycle sequencing reaction routinely included approximately100 or 200 ng isolated single-stranded DNA for A and C or G and Treactions, respectively. Double-stranded cycle sequencing reactionssimilarly contained approximately 200 or 400 ng of plasmid DNA isolatedusing either the standard alkaline lysis or the diatomaceousearth-modified alkaline lysis procedures. All reagents except templateDNA are added in one pipetting step from a premix of previouslyaliquoted stock solutions stored at −20° C. Reaction premixes areprepared by combining reaction buffer with the base-specific nucleotidemixes. Prior to use, the base-specific reaction premixes are thawed andcombined with diluted Taq DNA polymerase and the individual end-labeleduniversal primers to yield the final reaction mixes. Once the abovemixes are prepared, four aliquots of single or double-stranded DNA arepipetted into the bottom of each 0.2 ml thin-walled reaction tube,corresponding to the A, C, G, and T reactions, and then an aliquot ofthe respective reaction mixes is added to the side of each tube. Thesetubes are part of a 96-tube/retainer set tray in a microtiter plateformat, which fits into a Perkin Elmer Cetus Cycler 9600 (Foster City,Calif.). Strip caps are sealed onto the tube/retainer set and the plateis centrifuged briefly. The plate then is placed in the cycler whoseheat block had been preheated to 95° C., and the cycling programimmediately started. The cycling protocol consists of 15-30 cycles of:95° C. denaturation; 55° C. annealing; 72° C. extension; 95° C.denaturation; 72° C. extension; 95° C. denaturation, and 72° C.extension, linked to a 4° C. final soak file.

At this stage, the reactions may be frozen and stored at −20° C. for upto several days. Prior to pooling and precipitation, the plate iscentriftiged briefly to reclaim condensation. The primer andbase-specific reactions are pooled into ethanol, and the precipitatedDNA is collected by centrifugation and dried. These sequencing reactionscould be stored for several days at −20° C.

The protocol for the sequencing reactions is as follows. For A and Creactions, 1 μl, and for G and T reactions, 2 μl of each DNA sample (100ng/ul for M13 templates and 200 ng/ul for pUC templates) are pipettedinto the bottom of the 0.2 ml thin-walled reaction tubes. AmpliTaqpolymerase (N801-0060) is from Perkin-Elmer Cetus (Foster City, Calif.).

A mix of 30 μl AmpliTaq (5U/μl), 30 μl 5× Taq reaction buffer, 130 μlddH20, and 190 μl diluted Taq for 24 clones is prepared.

A, C, G, and T base specific mixes are prepared by adding base-specificprimer and diluted Taq to each of the base specific nucleotide/bufferpremixes:

A,C/G,T  60/120 μl 5X Taq cycle sequencing mix  30/60  μl diluted Taqpolymerase  30/60  μl respective fluorescent end-labeled primer 120/240μl

B. Taq-polymerase Catalyzed Cycle Sequencing Using MW-identifier-labeledTerminator Reactions

One problem in DNA cycle sequencing is that when primers are used thereaction conditions are such that the nested fragment set distributionis highly dependent upon the template concentration in the reaction mix.It has been recently observed that the nested fragment set distributionfor the DNA cycle sequencing reactions using the labeled terminators ismuch less sensitive to DNA concentration than that obtained with thelabeled primer reactions as described above. In addition, the terminatorreactions require only one reaction tube per template while the labeledprimer reactions require one reaction tube for each of the fourterminators. The protocol described below is easily interfaced with the96 well template isolation and 96 well reaction clean-up procedures alsodescribed herein.

Place 0.5 μg of single-stranded or 1 μg of double-stranded DNA in 0.2 mlPCR tubes. Add 1 μl (for single stranded templates) or 4 μl (fordouble-stranded templates) of 0.8 μM primer and 9.5 μl of ABI suppliedpremix to each tube, and bring the final volume to 20 μl with ddH₂O.Centrifuge briefly and cycle as usual using the terminator program asdescribed by the manufacturer (i.e., preheat at 96° C. followed by 25cycles of 96° C. for 15 seconds, 50° C. for 1 second, 60° C. for 4minutes, and then link to a 4° C. hold). Proceed with the spin columnpurification using either the Centri-Sep columns (Amicon, Beverly,Mass.) or G-50 microtiter plate procedures given below.

C. Terminator Reaction Clean-Up via Centri-Sep Columns

A column is prepared by gently tapping the column to cause the gelmaterial to settle to the bottom of the column. The column stopper isremoved and 0.75 ml dH₂O is added. Stopper the column and invert itseveral times to mix. Allow the gel to hydrate for at least 30 minutesat room temperature. Columns can be stored for a few days at 4° C. Allowcolumns that have been stored at 4° C. to warm to room temperaturebefore use. Remove any air bubbles by inverting the column and allowingthe gel to settle. Remove the upper-end cap first and then remove thelower-end cap. Allow the column to drain completely, by gravity. (Note:If flow does not begin immediately apply gentle pressure to the columnwith a pipet bulb.) Insert the column into the wash tube provided. Spinin a variable-speed microcentrifuge at 1300×g for 2 minutes to removethe fluid. Remove the column from the wash tube and insert it into asample collection tube. Carefully remove the reaction mixture (20 μl)and load it on top of the gel material. If the samples were incubated ina cycling instrument that required overlaying with oil, carefully removethe reaction from beneath the oil. Avoid picking up oil with the sample,although small amounts of oil (<1 μl) in the sample will not affectresults. Oil at the end of the pipet tip containing the sample can beremoved by touching the tip carefully on a clean surface (e.g., thereaction tube). Use each column only once. Spin in a variable-speedmicrocentrifuge with a fixed angle rotor, placing the column in the sameorientation as it was in for the first spin. Dry the sample in a vacuumcentrifuge. Do not apply heat or over dry. If desired, reactions can beprecipitated with ethanol.

D. Terminator Reaction Clean-Up via Sephadex G-50 Filled MicrotiterFormat Filter Plates

Sephadex (Pharmacia, Piscataway, N.J.) settles out; therefore, you mustresuspend before adding to the plate and also after filling every 8 to10 wells. Add 400 μl of mixed Sephadex G-50 to each well of microtiterfilter plate. Place microtiter filter plate on top of a microtiter plateto collect water and tape sides so they do not fly apart duringcentrifugation. Spin at 1500 rpm for 2 minutes. Discard water that hasbeen collected in the microtiter plate. Place the microtiter filterplate on top of a microtiter plate to collect water and tape sides sothey do not fly apart during centrifugation. Add an additional 100-200μl of Sephadex G-50 to fill the microtiter plate wells. Spin at 1500 rpmfor 2 minutes. Discard water that has been collected in the microtiterplate. Place the microtiter filter plate on top of a microtiter platewith tubes to collect water and tape sides so they do not fly apartduring centrifugation. Add 20 μl terminator reaction to each SephadexG-50 containing wells. Spin at 1500 rpm for 2 minutes. Place thecollected effluent in a Speed-Vac for approximately 1-2 hours.

Sequenase™ (UBS, Cleveland Ohio) catalyzed sequencing with labeledterminators. Single-stranded terminator reactions require approximately2 μg of phenol extracted M13-based template DNA. The DNA is denaturedand the primer annealed by incubating DNA, primer, and buffer at 65° C.After the reaction cooled to room temperature,alpha-thio-deoxynucleotides, labeled terminators, and diluted SequenaseTM DNA polymerase are added and the mixture is incubated at 37° C. Thereaction is stopped by adding ammonium acetate and ethanol, and the DNAfragments are precipitated and dried. To aid in the removal ofunincorporated terminators, the DNA pellet is rinsed twice with ethanol.The dried sequencing reactions could be stored up to several days at−20° C.

Double-stranded terminator reactions required approximately 5 μg ofdiatomaceous earth modified-alkaline lysis midi-prep purified plasmidDNA. The double-stranded DNA is denatured by incubating the DNA insodium hydroxide at 65° C., and after incubation, primer is added andthe reaction is neutralized by adding an acid-buffer. Reaction buffer,alpha-thio-deoxynucleotides, labeled dye-terminators, and dilutedSequenase TM DNA polymerase then are added and the reaction is incubatedat 37° C. Ammonium acetate is added to stop the reaction and the DNAfragments are precipitated, rinsed, dried, and stored.

For Single-stranded reactions:

Add the following to a 1.5 ml microcentrifuge tube:

4 μl ss DNA (2 μg) 4 μl 0.8 μM primer 2 μl 10x MOPS buffer 2 μl 10xMn²⁺/isocitrate buffer

To denature the DNA and anneal the primer, incubate the reaction at 65°C.-70° C. for 5 minutes. Allow the reaction to cool at room temperaturefor 15 minutes, and then briefly centrifuge to reclaim condensation. Toeach reaction, add the following reagents and incubate for 10 minutes at37° C.

7 μl ABI terminator mix (Catalogue No. 401489) 2 μl diluted Sequenase TM(3.25 U/μl) 1 μl 2 mM α-S dNTPs

The undiluted Sequenase TM (Catalogue No. 70775, United StatesBiochemicals, Cleveland, Ohio) is 13 U/μl and is diluted 1:4 with USBdilution buffer prior to use. Add 20 μl 9.5 M ammonium acetate and 100μl 95% ethanol to stop the reaction and mix.

Precipitate the DNA in an ice-water bath for 10 minutes. Centrifuge for15 minutes at 10,000×g in a microcentrifuge at 4° C. Carefully decantthe supernatant, and rinse the pellet by adding 300 μl of 70-80%ethanol. Mix and centrifuge again for 15 minutes and carefully decantthe supernatant.

Repeat the rinse step to insure efficient removal of the unincorporatedterminators. Dry the DNA for 5-10 minutes (or until dry) in theSpeed-Vac, and store the dried reactions at −20° C.

For double-stranded reactions:

Add the following to a 1.5 ml microcentrifuge tube:

5 μl ds DNA (5 μg) 4 μl 1 N NaOH 3 μl ddH₂O

Incubate the reaction at 65° C.-70° C. for 5 minutes, and then brieflycentrifuge to reclaim condensation. Add the following reagents to eachreaction, vortex, and briefly centrifuge:

3 μl 8 μM primer 9 μl ddH₂O 4 μl MOPS-Acid buffer

To each reaction, add the following reagents and incubate for 10 minutesat 37° C.

4 μl 10X Mn²⁺/isocitrate buffer 6 μl ABI terminator mi 2 μl dilutedSequenase TM (3.25 U/μl) 1 μl 2 mM [alpha]-S-dNTPS

The undiluted SEQUENASE™ from United States Biochemicals is 13 U/μl andshould be diluted 1:4 with USB dilution buffer prior to use. Add 60 μl 8M ammonium acetate and 300 μl 95% ethanol to stop the reaction andvortex. Precipitate the DNA in an ice-water bath for 10 minutes.Centrifuge for 15 minutes at 10,000×g in a microcentrifuge at 4° C.Carefully decant the supernatant, and rinse the pellet by adding 300 μlof 80% ethanol. Mix the sample and centrifuge again for 15 minutes, andcarefully decant the supernatant. Repeat the rinse step to insureefficient removal of the unincorporated terminators. Dry the DNA for5-10 minutes (or until dry) in the Speed-Vac.

E. Sequence Gel Preparation, Pre-electrophoresis Sample Loading,Electrophoresis, Data Collection, and Analysis on the ABI 373A DNASequencer

Polyacrylamide gels for DNA sequencing are prepared as described above,except that the gel mix is filtered prior to polymerization. Glassplates are carefully cleaned with hot water, distilled water, andethanol to remove potential fluorescent contaminants prior to taping.Denaturing 6% polyacrylamide gels are poured into 0.3 mm×89 cm×52 cmtaped plates and fitted with a 36 well comb. After polymerization, thetape and the comb are removed from the gel and the outer surfaces of theglass plates are cleaned with hot water, and rinsed with distilled waterand ethanol. The gel is assembled into an ABI sequencer, and the checkedby laser-scanning. If baseline alterations are observed on the ABIassociated Macintosh computer display, the plates are recleaned.Subsequently, the buffer wells are attached, electrophoresis buffer isadded, and the gel is pre-electrophoresed for 10-30 minutes at 30 W.Prior to sample loading, the pooled and dried reaction products areresuspended in formamide/EDTA loading buffer by vortexing and thenheated at 90° C. A sample sheet is created within the ABI datacollection software on the Macintosh computer which indicates the numberof samples loaded and the fluorescent-labeled mobility file to use forsequence data processing. After cleaning the sample wells with asyringe, the odd-numbered sequencing reactions are loaded into therespective wells using a micropipettor equipped with a flat-tippedgel-loading tip. The gel is then electrophoresed for 5 minutes beforethe wells are cleaned again and the even numbered samples are loaded.The filter wheel used for dye-primers and dye-terminators is specifiedon the ABI 373A CPU. Typically electrophoresis and data collection arefor 10 hours at 30 W on the ABI 373A that is fitted with aheat-distributing aluminum plate. After data collection, an image fileis created by the ABI software that relates the fluorescent signaldetected to the corresponding scan number. The software then determinesthe sample lane positions based on the signal intensities. After thelanes are tracked, the cross-section of data for each lane are extractedand processed by baseline subtraction, mobility calculation, spectraldeconvolution, and time correction. After processing, the sequence datafiles are transferred to a SPARCstation 2 using NFS Share.

Protocol: prepare 8 M urea, 4.75% polyacrylamide gels, as describedabove, using a 36-well comb. Prior to loading, clean the outer surfaceof the gel plates. Assemble the gel plates into an ABI 373A DNASequencer (Foster City, Calif.) so that the lower scan (usually theblue) line corresponds to an intensity value of 800-1000 as displayed onthe computer data collection window. If the baseline of four-color scanlines is not flat, reclean the glass plates. Affix the aluminum heatdistribution plate. Pre-electrophorese the gel for 10-30 minutes.Prepare the samples for loading. Add 3 μl of FE to the bottom of eachtube, vortex, heat at 90° C. for 3 minutes, and centrifuge to reclaimcondensation. Flush the sample wells with electrophoresis buffer using asyringe. Using flat-tipped gel loading pipette tips. load eachodd-numbered sample. Pre-electrophorese the gel for at least 5 minutes,flush the wells again, and then load each even-numbered sample. Beginthe electrophoresis (30 W for 10 hours). After data collection, the ABIsoftware will automatically open the data analysis software, which willcreate the imaged gel file, extract the data for each sample lane, andprocess the data.

F. Double-stranded Sequencing of cDNA Clones Containing Long Poly(A)Tails Using Anchored Poly(dT) Primers

Double-stranded templates of cDNAs containing long poly(A) tracts aredifficult to sequence with vector primers which anneal downstream of thepoly(A) tail. Sequencing with these primers results in a long poly(T)ladder followed by a sequence which may be difficult to read. Tocircumvent this problem, three primers which contain (dT)₁₇ and either(dA) or (dC) or (dG) at the 3′ end were designed to ‘anchor’ the primersand allow sequencing of the region immediately upstream of the poly(A)region. Using this protocol, over 300 bp of readable sequence could beobtained. The sequence of the opposite strand of these cDNAs wasdetermined using insert-specific primers upstream of the poly(A) region.The ability to directly obtain sequence immediately upstream from thepoly(A) tail of cDNAs should be of particular importance to large scaleefforts to generate sequence-tagged sites (STSs) from cDNAs.

The protocol is as follows. Synthesize anchored poly (dT)₁₇ with anchorsof (dA) or (dC) or (dG) at the 3′ end on a DNA synthesizer and use afterpurification on Oligonucleotide Purification Cartridges (Amicon,Beverly, Mass.). For sequencing with anchored primers, denature 5-10 μgof plasmid DNA in a total volume of 50 μl containing 0.2 M sodiumhydroxide and 0.16 mM EDTA by incubation at 65° C. for 10 minutes. Addthe three poly(dT) anchored primers (2 pmol of each) and immediatelyplace the mixture on ice. Neutralize the solution by adding 5 ml of 5 Mammonium acetate, pH 7.0.

Precipitate the DNA by adding 150 μl of cold 95% ethanol and wash thepellet twice with cold 70% ethanol. Dry the pellet for 5 minutes andthen resuspend in MOPS buffer. Anneal the primers by heating thesolution for 2 minutes at 65° C. followed by slow cooling to roomtemperature for 15-30 minutes. Perform sequencing reactions, usingmodified T7 DNA polymerase and α-[³²P]dATP (>1000 Ci/mmole) using theprotocol described above.

G. cDNA Sequencing Based on PCR and Random Shotgun Cloning

The following is a method for sequencing cloned cDNAs based on PCRamplification, random shotgun cloning, and automated fluorescentsequencing. This PCR-based approach uses a primer pair between the usual“universal” forward and reverse priming sites and the multiple cloningsites of the Stratagene Bluescript vector. These two PCR primers, withthe sequence 5′-TCGAGGTCGACGGTATCG-3′ (Seq. ID No. 15) for the forwardor −16 bs primer and 5′-GCCGCTCTAGAACTAG TG-3′ (Seq. ID No. 16) for thereverse or +19 bs primer, may be used to amplify sufficient quantitiesof cDNA inserts in the 1.2 to 3.4 kb size range so that the randomshotgun sequencing approach described below could be implemented.

The following is the protocol. Incubate four 100 μl PCR reactions, eachcontaining approximately 100 ng of plasmid DNA, 100 pmoles of eachprimer, 50 mM KCl, 10 mM Tris-HCl pH 8.5, 1.5 mM MgCl₂, 0.2 mM of eachdNTP, and 5 units of PE-Cetus Amplitaq in 0.5 ml snap cap tubes for 25cycles of 95° C. for 1 minute, 55° C. for 1 minute and 72° C. for 2minutes in a PE-Cetus 48 tube DNA Thermal Cycler. After pooling the fourreactions, the aqueous solution containing the PCR product is placed inan nebulizer, brought to 2.0 ml by adding approximately 0.5 to 1.0 ml ofglycerol, and equilibrated at −20° C. by placing it in either anisopropyl alcohol/dry ice or saturated aqueous NaCl/dry ice bath for 10minutes. The sample is nebulized at −20° C. by applying 25-30 psinitrogen pressure for 2.5 min. Following ethanol precipitation toconcentrate the sheared PCR product, the fragments were blunt ended andphosphorylated by incubation with the Klenow fragment of E. coli DNApolymerase and T4 polynucleotide kinase as described previously.Fragments in the 0.4 to 0.7 kb range were obtained by elution from a lowmelting agarose gel.

Example 18 SEPARATION OF DNA FRAGMENTS

Instrumentation

The separation of DNA fragments can be performed using an HPLC systemassembled from several standard components. These components include aminimum of two high pressure pumps which pump solvent through a highpressure gradient mixer, an injector, HPLC column, and a detector. Theinjector is an automated, programmable autosampler capable of storingtypically between eighty and one hundred samples at or below ambienttemperatures to maintain the stability of the sample components. Theautoinjector also is capable of making uL size injections in areproducible manner completely unattended. The HPLC column is containedin a heated column compartment capable of holding a defined temperatureto within 0.1° C. The column used in the examples below was purchasedfrom SeraSep (San Jose, Calif.) under the name DNASep. This column is a55×4.6 mm column with a 2.2 um non-porous polystyrenedivinylbenzenecopolymer particle alkylated with C18. The packing material is stablewithin a pH range of 2-12 and tolerates temperatures as high as 70° C.Detection of analyte was performed using a single or multiple wavelengthUV detector or diode array detector.

Methods

The methods applied in this example for separation of DNA fragments useion-pair chromatography, a form of chromatography in which ions insolution can be paired or neutralized and separated as an ion pair on areversed phase column. The lipophilic character and the concentration ofthe counterion determine the degree of retention of the analyte. In thecase of a DNA molecule the lipophilic, cationic buffer component pairswith anionic phosphate groups of the DNA backbone. The buffer componentsalso interact with the alkyl groups of the stationary phase. The pairedDNA then elutes according to size as the mobile phase is madeprogressively more organic with increasing concentration ofacetonitrile. Evaluation of the suitability of various amine salts wasevaluated using enzymatic digests of plasmids or commercially availableDNA ladders. The range of acetonitrile required to elute the DNA as wellas the temperature of the column compartment varied with each bufferevaluated.

Buffers

The buffers evaluated for their ion-pairing capability were preparedfrom stock solutions. In order to keep the concentration of ion-pairreagent the same throughout the gradient, the ion-pair reagent was addedto both the water and the acetonitrile mobile phases. The column wasequilibrated with a new mobile phase for approximately 18 hours at aflow rate of 50 ul/minute before attempting any separation. Once amobile phase had been evaluated, it was removed and the column cleanedwith a flush of 800 mL 0.1% formic acid in 50% acetonitrile, followed bya flush with 800 mL 0.1% acetic acid in 50% acetonitrile beforeequilibration with a new mobile phase.

A. N,N-Dimethyloctylammonium trifluoroacetate

A stock solution of lmolar dimethyloctylammonium trifluoroacetate wasprepared by mixing one half of an equivalent of trifluoroacetic acid inan appropriate volume of water and slowly adding one equivalent ofnn-Dimethyloctylamine. The pH of this stock solution is 7. The stocksolution was diluted with an appropriate volume of water or acetonitrileto working concentration.

B. N,N-Dimethylheptylammonium acetate

A stock solution of Imolar dimethylheptylammonium acetate was preparedby mixing one equivalent of glacial acetic acid in an appropriate volumeof water and slowly adding one equivalent of nn-Dimethylheptylamine. ThepH of this stock solution is 6.6. The stock solution was diluted with anappropriate amount of water or acetonitrile to working concentration.

C. N,N-Dimethylhexylammonium acetate

A stock solution of 1 molar dimethylhexylammonium acetate was preparedby mixing one equivalent of glacial acetic acid in an appropriate volumeof water and slowly adding one equivalent of nn-Dimethylhexylamine. ThepH of this stock solution is 6.5. The stock solution was diluted with anappropriate volume of water or acetonitrile to working concentration.

D. N,N-Dimethylbutylammonium acetate

A stock solution of Imolar dimethylbutylammonium acetate was prepared bymixing one equivalent of glacial acetic acid in an appropriate volume ofwater and slowly adding one equivalent of nn-Dimethylbutylamine. The pHof the stock solution is 6.9. The stock solution was diluted with anappropriate volume of water or acetonitrile to working concentration.

E. N,N-Dimethylisopropylammonium acetate

A stock solution of 1 molar dimethylisopropylammonium acetate wasprepared by mixing one equivalent of glacial acetic acid in anappropriate volume of water and slowly adding one equivalent ofnn-Dimethylisopropylamine. The pH of the stock solution is 6.9. Thestock solution was diluted with an appropriate volume of water oracetonitrile to working concentration.

F. N,N-Dimethylcyclohexylammonium acetate

A stock solution of 1 molar dimethylcyclohexylammonium acetate wasprepared by mixing one equivalent of glacial acetic acid in appropriatevolume of water and slowly adding one equivalent ofnn-Dimethylcyclohexylamine. The pH of the stock solution is 6.5. Thestock solution was diluted with an appropriate volume of water oracetonitrile to working concentration.

G. Methylpiperidine acetate

A stock solution of 1 molar methylpiperidine acetate was prepared bymixing one equivalent of glacial acetic acid in an appropriate volume ofwater and slowly adding one equivalent of 1-methylpiperidine. The pH ofthe solution is 7. The stock solution was diluted with an appropriatevolume of water or acetonitrile to working concentration.

H. Methylpyrrolidine acetate

A stock solution of 1 molar piperidine acetate was prepared by mixingone equivalent of glacial acetic acid in an appropriate volume of waterand slowly adding one equivalent 1-methylpyrrolidine. The pH of thestock solution is 7. The stock solution was diluted in an appropriatevolume of water or acetonitrile to working concentration.

I. Triethylammonium acetate

A stock solution of 2 molar triethylammonium acetate pH 7.0 waspurchased from Glenn Research Sterling, Va. The stock solution wasdiluted in an appropriate volume of water or acetonitrile to workingconcentration.

Example 19

In this Example (19), all reactions were conducted in foil-coveredflasks. The sequence of reactions A→F described in this Example isillustrated in FIGS. 17A and 17B. Compound numbers as set forth in thisExample refer to the compounds of the same number in FIGS. 17A and 17B.

A. To a solution of ANP linker (compound 1, 11.2 mmol) anddiisopropylethylamine (22.4 mmol) in CHCl₃ (60 ml) was added allylbromide (22.4 mmol). The reaction mixture was refluxed for 3 hours,stirred at room temperature for 18 hours, diluted with CHCl₃ (200 ml),and washed with 1.0 M HCl (2×150 ml) and H₂O (2×150 ml). The organicextracts were dried (MgSO₄) and the solvent evaporated to give compound2 as a yellow solid.

To a mixture of compound 2 in CH₂Cl₂ (70 ml), tris (2-aminoethyl) amine(50 ml) was added and the reaction mixture stirred at room temperaturefor 18 hours. The reaction was diluted with CH₂Cl₂ (150 ml) and washedwith pH 6.0 phosphate buffer (2×150 ml). The organic extracts were dried(MgSO₄) and the solvent evaporated. The residue was subjected to columnchromatography (hexane/EtOAc) to give 1.63 g (58%/o) of compound 3: ¹HNMR (DMSO-d₆): δ 7.85 (dd, 2H), 7.70 (t, 1H), 7.43 (t, 1H), 5.85 (m,1H), 5.20 (q, 2H), 4.58 (q, 1H), 4.50 (d, 2H), 2.70 (m, 2H), 2.20 (br s,2H).

B. To a solution of Boc-5-aminopentanoic acid (1.09 mmol) and NMM (3.27mmol) in dry DMF (6 ml), was added HATU (1.14 mmol) and the reactionmixture stirred at room temperature for 0.5 hours. A solution ofcompound 3 (1.20 mmol) in dry DMF (1 ml) was added and the reactionmixture stirred at room temperature for 18 hours. The reaction wasdiluted with EtOAc (50 ml) and washed with 1.0 M HCl (2×50 ml) and D.I.H₂O (2×50 ml). The organic extracts were dried (MgSO₄) and evaporated todryness. The residue was subjected to column chromatography to give 420mg (91%) of compound 4: ¹H NMR (DMSO-d₆): δ 8.65 (d, 1H), 7.88 (d, 1H),7.65 (m, 2H), 7.48 (t, 1H), 6.73 (br s, 1H), 5.85 (m, 1H), 5.55 (m, 1H),5.23 (q, 2H), 4.55 (d, 2H), 2.80 (m, 2H), 2.05 (t, 2H), 1.33 (s, 9H),1.20-1.30 (m, 4H).

C. A solution of compound 4 (0.9 mmol) in HCl•1,4-dioxane (20 mmol) wasstirred at room temperature for 2 hours. The reaction mixture wasconcentrated, dissolved in MeOH and toluene, and concentrated again (5×5ml) to give 398 mg (quantitative) of the compound 5: ¹H NMR (DMSO-d₆): δ8.75 (d, 1H), 7.88 (d, 1H), 7.65 (m, 2H),7.51 (t, 1H), 7.22 (m, 2H),5.85(m, 1H), 5.57 (m, 1H), 5.23 (q, 2H), 4.55 (d, 2H), 2.80 (m, 2H), 2.71(m, 2H), 2.07 (s, 2H), 1.40-1.48 (br s, 4 H).

D. To a solution of compound 21 (0.48 mmol, prepared according toExample 21) and NMM (1.44 mmol) in dry DMF (3 ml), was added HATU (0.50mmol) and the reaction mixture stirred at room temperature for 0.5hours. A solution of compound 5 (0.51 mmol) in dry DMF (3 ml) was addedand the reaction stirred at room temperature for 18 hours. The reactionmixture was diluted with EtOAc (75 ml) and washed with 5% Na₂CO₃ (3×50ml). The organic extracts were dried (MgSO₄) and the solvent evaporatedto give 281 mg (78%) of compound 6: ¹H NMR (DMSO-d₆): δ 8.65 (d, 1H),8.17 (d, 1H), 7.82-7.95 (m, 4H), 7.68 (m, 3H), 7.50 (t, 1H), 6.92 (d,1H), 5.85 (m, 1H), 5.57 (m, 1H), 5.20 (q, 2H), 4.55 (d, 2H), 4.30 (q,1H), 4.05 (q, 2H), 2.95 (m, 4H), 2.80 (m, 2H), 2.72 (m, 2H), 2.05 (s,3H), 2.01 (t, 2H), 1.58-1.77 (m, 3H), 1.50 (m, 4H), 1.30 (q, 3H),1.17-1.40 (m, 9H).

E. To a mixture of compound 6 (0.36 mmol) in THF (4 ml), was added 1 MNaOH (1 mmol) and the reaction stirred at room temperature for 2 hours.The reaction mixture was acidified to pH 7.0 with 1.0 M HCl (1 ml) andthe solvent evaporated to give compound 7 (quantitative): ¹H NMR(DMSO-d₆): δ 8.65 (d, 1H), 8.17 (d, 1H), 7.82-7.95 (m, 4H), 7.68 (m,3H), 7.50 (t, 1H), 6.92 (d, 1H), 5.52 (m, 1H), 4.30 (q, 1H), 4.05 (q,2H), 2.95 (m, 4H), 2.80 (m, 2H), 2.72 (m, 2H), 2.05 (s, 3H), 2.01 (t,2H), 1.58-1.77 (m, 3H), 1.50 (m, 4H), 1.30 (q, 3H), 1.17-1.40 (m, 9H).

F. To a solution of compound 7 (0.04 mmol) and NMM (0.12 mmol) in dryDMF (0.4 ml), was added HATU (0.044 mmol) and the reaction stirred atroom temperature for 0.5 hours. Allylamine (0.12 mmol) was added and thereaction mixture stirred at room temperature for 5 hours. The reactionmixture was diluted with EtOAc (15 ml) and washed with 5% Na₂CO₃ (3×10ml). The organic extracts were dried (MgSO₄) and the solvent evaporatedto yield 15 mg (49%) of compound 8: ¹H NMR (DMSO-d₆) δ 8.49 (d, 1H),8.17 (d, 1H), 7.82-7.95 (m, 4H), 7.68 (m, 3H), 7.50 (t, 1H), 6.92 (d,1H), 5.72 (m, 1H), 5.50 (m, 1H), 5.03 (q, 2H), 4.37 (d, 2H), 4.30 (q,1H), 4.05 (q, 2H), 2.95 (m, 4H), 2.80 (m, 2H), 2.72 (m, 2H), 2.05 (s,3H), 2.01 (t, 2H), 1.58-1.77 (m, 3H), 1.50 (m, 4H), 1.30 (q, 3H),1.17-1.40 (m, 9H).

Example 20

The sequence of reactions A→G as described in this Example 20 isillustrated in FIGS. 18A and 18B. Compound numbers as set forth in thisExample refer to the compounds of the same number in FIGS. 18A and 18B.

A. To a solution of Fmoc-Lys(Boc)—OH (compound 9, 33.8 mmol) in CHCl₃(200 ml), was added diisopropylethylamine (67.5 mmol) and allyl bromide(67.5 mmol). The reaction mixture was refluxed for 6 hours, stirred atroom temperature for 16 hours, diluted with CHCl₃, washed with 1.0 M HCl(2×150 ml), saturated NaHCO₃ (1×150 ml) and D.I. H₂O (2×150 ml). Theorganic extracts were dried (MgSO₄) and the solvent evaporated to yieldcompound 10.

To a solution of compound 10 in CHCl₃ (90 ml), was added pyrrolidine (10eq.) and the reaction was stirred at room temperature for 2.5 hours. Thereaction mixture was diluted with CHCl₃ (150 ml) and washed withsaturated NaHCO₃ (3×250 ml). The organic extracts were dried (MgSO₄) andthe solvent evaporated. The residue Nwas subjected to columnchromatography (EtOAc/MeOH) to give 6.52 g (67%) of compound 11: ¹H NMR(CDCl₃): δ 5.90 (m, 1H), 5.27 (m, 2H), 4.60 (d, 2H), 3.48 (t, 1H), 3.10(d, 2H), 1.40-1.78 (m, 9H), 1.40 (s, 9H).

B. To a solution of N-methylisonipecotic acid (1.60 mmol) and N-methylmorpholine (4.80 mmol) in dry DMF (5 ml), was added HATU (1.67 mmol).After 0.5 hours, a solution of compound 11 (1.75 mmol) in dry DMF (2 ml)was added and the reaction mixture stirred at room temperature for 18hours. The reaction mixture was diluted with CH₂CL₂ (60 ml) and washedwith saturated Na₂CO₃ (3×40 ml). The organic extracts were dried (MgSO₄)and the solvent evaporated. The residue was subjected to columnchromatography (CH₂Cl₂/MeOH/triethylamine) to give 580 mg (88%) ofcompound 12: ¹H NMR (DMSO): δ 8.12 (d, 1H), 6.77 (t, 1H), 5.90 (m, 1H),5.27 (m, 2H), 4.53 (d, 2H), 4.18 (m, 1H),2.62-2.90 (m, 5H), 2.13 (s,3H), 1.85 (m, 2H), 1.57 (m, 5H), 1.35 (s, 9H), 1.00 (t, 2H).

C. A mixture of compound 12 (1.39 mmol) in HCl•1,4-dioxane (20 mmol) wasstirred at room temperature for 4 hours. The reaction mixture wasconcentrated, dissolved in MeOH, coevaporated with toluene (5×5 ml) togive 527 mg (quantitative) of compound 13: ¹H NMR (DMSO-d₆): δ 8.12 (d,1H), 6.77 (t, 1H), 5.90 (m, 1H), 5.27 (m, 2H), 4.53 (d, 2H), 4.18 (m,1H), 2.65-3.00 (m, 8H), 2.23 (s, 3H), 1.85 (m, 2H), 1.57 (m, 5H), 1.00(t, 2H).

D. To a solution of 4-ethoxybenzoic acid (1 eq.) in dry DMF, is addedNMM (3 eq.) and HATU (1.05 eq.). After 0.5 hours, a solution of compound13 in dry DMF is added. After the completion of the reaction and basicworkup, the compound 14 is isolated and purified.

E. To a solution of compound 14 in THF, is added 1N NaOH and thereaction mixture stirred at room temperature. After the completion ofthe reaction and acidification, the compound 15 is isolated.

F. To a solution of compoumd 15 (1 eq.) in dry DMF, is added NIMM (3eq.) and HATU (1.05 eq.). After 0.5 hours, a solution of compound 21(ANP-allyl ester, prepared according to Example 21) in dry DMF is added.After the completion of the reaction and basic workup, the titlecompound 16 is isolated and purified.

G. To a solution of compound 16 in THF, is added 1N NaOH and thereaction mixture stirred at room temperature. After the completion ofthe reaction and acidification, the compound 17 is isolated.

Example 21

The sequence of reaction A through D as described in this Example 21 isillustrated in FIG. 19. Compound numbers as set forth in this Example,as well as Examples 19 and 20, refer to the compounds of the same numberin FIG. 19.

A. To a solution of 4-ethoxybenzoic acid (7.82 mmol) and N-methylmorpholine (20.4 mmol) in CH₂Cl₂ (10 ml), was added HATU (7.14 mmol).After 0.25 hours, a solution of compound 11 (6.8 mmol) in CH₂Cl₂ (6 ml)was added and the reaction mixture stirred at room temperature for 18hours. The reaction was diluted with CH₂Cl₂ (150 ml) and washed with 1.0M HCl (3×50 ml) and saturated NaHCO₃ (3×50 ml). The organic extractswere dried (MgSO₄) and the solvent evaporated. The residue was subjectedto column chromatography (CH₂Cl₂/MeOH) to give 2.42 g (82%) of compound18: ¹H NMR (CDCl): δ 7.78 (d, 2H), 6.91 (d, 2H), 6.88 (d, 1H), 5.83-5.98(m, 1H), 5.21-5.38 (m, 2H), 4.80 (q, 1H), 4.66 (d, 2H), 4.06 (q, 2H),3.11 (q, 2H), 1.90-2.04 (m, 1H), 1.68-1.87 (m, 1H), 1.39 (t, 3H), 1.34(s, 9H), 1.32-1.58 (m, 4H).

B. A mixture of compound 18 (5.5 mmol) in HCl•1,4-dioxane (14.3 mmol)was stirred at room temperature for 1 hour. The reaction mixture wasconcentrated, dissolved in MeOH, azeotroped with toluene, andconcentrated again (5×5 ml) to give a quantitative yield of compound 19.

C. To a solution of N-methylisonipecotic acid (6.21 mmol) in dry DMF (15mL), was added NMM (21.6 mmol) and HATU (5.67 mmol). After 0.5 hours, asolution of compound 19 (5.4 mmol) in dry DMF (10 ml) was added and thereaction stirred at room temperature for 18 hours. The reaction mixturewas brought to pH 12 with 1N NaOH (20 ml) and extracted with CHCl₃(2×200 ml). The organic extracts were dried (MgSO₄) and the solventevaporated to give 2.2 g (89%) of compound 20: ¹H NMR (DMSO-d₆): δ 8.52(d, 1H), 7.84 (d, 2H), 7.72 (t, 1H), 6.95 (d, 2H), 5.80-5.95 (m, 1H),5.18-5.31 (dd, 2H), 4.58 (d, 2H), 4.37 (q, 1H), 4.08 (q, 2H), 3.01 (d,2H), 2.08 (s, 3H), 1.95 (m, 1H), 1.63-1.82 (m, 4H), 1.51 (m, 4H), 1.32(t,3H), 1.22-1.41 (m, 6H).

D. To a solution of compound 20 (4.4 mmol) in THF (10 ml), is added 1NNaOH (4.4 mmol) and the reaction mixture stirred at room temperature for1 hour. The reaction was concentrated, dissolved in THF/toluene (2×5ml), concentrated, dissolved in CH₂Cl₂/toluene (1×5 ml) and concentratedagain to give a quantitative yield of compound 21: ¹H NMR (DMSO-d₆): δ7.76 (d, 2H), 6.96 (d, 2H), 4.04 (q, 2H), 3.97 (d, 1H), 2.97 (d, 2H),2.64 (d, 2H), 2.08 (s, 3H), 1.95 (m, 1H), 1.58-1.79 (m, 4H), 1.44 (m,6H), 1.30 (t, 3H), 1.11-1.35 (m, 4H).

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

16 18 base pairs nucleic acid single linear not provided 1 TGTAAAACGACGGCCAGT 18 19 base pairs nucleic acid single linear not provided 2TGTAAAACGA CGGCCAGTA 19 20 base pairs nucleic acid single linear notprovided 3 TGTAAAACGA CGGCCAGTAT 20 21 base pairs nucleic acid singlelinear not provided 4 TGTAAAACGA CGGCCAGTAT G 21 22 base pairs nucleicacid single linear not provided 5 TGTAAAACGA CGGCCAGTAT GC 22 23 basepairs nucleic acid single linear not provided 6 TGTAAAACGA CGGCCAGTATGCA 23 24 base pairs nucleic acid single linear not provided 7TGTAAAACGA CGGCCAGTAT GCAT 24 25 base pairs nucleic acid single linearnot provided 8 TGTAAAACGA CGGCCAGTAT GCATG 25 18 base pairs nucleic acidsingle linear not provided 9 TGTAAAACGA CGGCCAGC 18 19 base pairsnucleic acid single linear not provided 10 TGTAAAACGA CGGCCAGCG 19 20base pairs nucleic acid single linear not provided 11 TGTAAAACGACGGCCAGCGT 20 21 base pairs nucleic acid single linear not provided 12TGTAAAACGA CGGCCAGCGT A 21 22 base pairs nucleic acid single linear notprovided 13 TGTAAAACGA CGGCCAGCGT AC 22 23 base pairs nucleic acidsingle linear not provided 14 TGTAAAACGA CGGCCAGCGT ACC 23 18 base pairsnucleic acid single linear not provided 15 TCGAGGTCGA CGGTATCG 18 18base pairs nucleic acid single linear not provided 16 GCCGCTCTAGAACTAGTG 18

We claim:
 1. A method for determining the sequence of a nucleic acidmolecule, comprising: (a) generating tagged nucleic acid fragments whichare complementary to a selected target nucleic acid molecule, wherein atag is an organic moiety that is correlative with a particularnucleotide and detectable by non-fluorescent spectrometry orpotentiometry; (b) separating the tagged fragments by sequential length;(c) cleaving the tags from the tagged fragments; and (d) detecting thetags by non-fluorescent spectrometry or potentiometry, and therefromdetermining the sequence of the nucleic acid molecule.
 2. The methodaccording to claim 1 wherein the detection of the tags is by massspectrometry, infrared spectrometry, ultraviolet spectrometry orpotentiostatic amperometry.
 3. The method according to claims 1 or 2wherein the tagged fragments are separated in step (b) by a methodselected from gel electrophoresis, capillary electrophoresis,micro-channel electrophoresis and HPLC.
 4. The method according toclaims 1 or 2 wherein the tagged fragments are cleaved in step (c) by amethod selected from oxidation, reduction, acid-labile, base-labile,enzymatic, electrochemical, thermal, thiol exchange and photolabilemethods.
 5. The method according to claim 2 wherein the tags aredetected by time-of-flight mass spectrometry, quadrupole massspectrometry, magnetic sector mass spectrometry or electric sector massspectrometry.
 6. The method according to claim 2 wherein the tags aredetected by coulometric detectors or amperometric detectors.
 7. Themethod according to claims 1 or 2 wherein the tagged nucleic acidfragments are generated in step (a) from a 5′ terminus to a 3′ terminus.8. The method according to claims 1 or 2 wherein step (a) generates morethan four of the tagged nucleic acid fragments and each tag is uniquefor a nucleic acid fragment.
 9. The method according to claims 1 or 2wherein steps (b), (c) and (d) are performed in a continuous manner. 10.The method according to claims 1 or 2 wherein steps (b), (c) and (d) areperformed in a continuous manner on a system.
 11. The method accordingto claims 1 or 2 wherein one or more of the steps is automated.
 12. Themethod according to claims 1 or 2 wherein the tagged fragments aregenerated from oligonucleotide primers that are conjugated to a tag atother than the 3′ end of the primer.
 13. The method according to claims1 or 2 wherein the tagged fragments are generated from taggeddideoxynucleotide terminators.
 14. The method according to claims 1 or 2wherein at least one tagged nucleic acid fragment is a compoundaccording to any one of claims 15 to
 33. 15. A compound of the formula:T^(ms)—L—X wherein, T^(ms) is an organic group detectable by massspectrometry, comprising carbon, at least one of hydrogen and fluoride,and optional atoms selected from oxygen, nitrogen, sulfur, phosphorusand iodine; L is an organic group which allows a uniqueT^(ms)-containing moiety to be cleaved from the remainder of thecompound, wherein the T^(ms)-containing moiety comprises a functionalgroup which supports a single ionized charge state when the compound issubjected to mass spectrometry and is selected from tertiary amine,quaternary amine and organic acid; X is a nucleic acid fragment attachedto L at other than the 3′ end of the nucleic acid fragment; with theprovisos that the compound is not bonded to a solid support nor has amass of less than 250 daltons.
 16. A compound according to claim 15wherein T^(ms) has a mass of from 15 to 10,000 daltons and a molecularformula of C₁₋₅₀₀N₀₋₁₀₀O₀₋₁₀₀S₀₋₁₀P₀₋₁₀H_(α)F_(β)I_(δ) wherein the sumof α, β and δ is sufficient to satisfy the otherwise unsatisfiedvalencies of the C, N, O, P and S atoms.
 17. A compound according toclaim 15 wherein T^(ms) and L are bonded together through a functionalgroup selected from amide, ester, ether, amine, sulfide, thioester,disulfide, thioether, urea, thiourea, carbamate, thiocarbamate, Schiffbase, reduced Schiff base, imine, oxime, hydrazone, phosphate,phosphonate, phosphoramide, phosphonamide, sulfonate, sulfonamide orcarbon-carbon bond.
 18. A compound according to claim 17 wherein thefunctional group is selected from amide, ester, amine, urea andcarbamate.
 19. A compound according to claim 15 wherein L is selectedfrom L^(hν), L^(acid), L^(base), L^([O]), L^([R]), L^(enz), L^(elc),L^(Δ) and L^(ss), where actinic radiation, acid, base, oxidation,reduction, enzyme, electrochemical, thermal and thiol exchange,respectively, cause the T^(ms)-containing moiety to be cleaved from theremainder of the molecule.
 20. A compound according to claim 19 whereinL^(hν) has the formula L¹—L²—L³, wherein L² is a molecular fragment thatabsorbs actinic radiation to promote the cleavage of T^(ms) from X, andL¹ and L³ are independently a direct bond or an organic moiety, where L¹separates L² from T^(ms) and L³ separates L² from X, and neither L¹ norL³ undergo bond cleavage when L² absorbs the actinic radiation.
 21. Acompound according to claim 20 wherein —L²—L³ has the formula:

with one carbon atom at positions a, b, c, d or e being substituted with—L³—X and optionally one or more of positions b, c, d or e beingsubstituted with alkyl, alkoxy, fluoride, chloride, hydroxyl,carboxylate or amide; and R¹ is hydrogen or hydrocarbyl.
 22. A compoundaccording to claim 21 wherein X is

and R² is nucleic acid fragment.
 23. A compound according to claim 20wherein L³ is selected from a direct bond, a hydrocarbylene,—O-hydrocarbylene, and hydrocarbylene-(O-hydrocarbylene)_(n)—H, and n isan integer ranging from 1 to
 10. 24. A compound according to claim 15wherein —L—X has the formula:

wherein one or more of positions b, c, d or e is substituted withhydrogen, alkyl, alkoxy, fluoride, chloride, hydroxyl, carboxylate oramide; R¹ is hydrogen or hydrocarbyl, and R² is a nucleic acid fragment.25. A compound according to claim 15 wherein T^(ms) has the formula:T²—(J—T³—)_(n)— T² is an organic moiety formed from carbon and one ormore of hydrogen, fluoride, iodide, oxygen, nitrogen, sulfur andphosphorus, having a mass of 15 to 500 daltons; T³ is an organic moietyformed from carbon and one or more of hydrogen, fluoride, iodide,oxygen, nitrogen, sulfur and phosphorus, having a mass of 50 to 1000daltons; J is a direct bond or a functional group selected from amide,ester, amine, sulfide, ether, thioester, disulfide, thioether, urea,thiourea, carbamate, thiocarbamate, Schiff base, reduced Schiff base,imine, oxime, hydrazone, phosphate, phosphonate, phosphoramide,phosphonamide, sulfonate, sulfonamide or carbon-carbon bond; and n is aninteger ranging from 1 to 50, and when n is greater than 1, each T³ andJ is independently selected.
 26. A compound according to claim 25wherein T² is selected from hydrocarbyl, hydrocarbyl-O-hydrocarbylene,hydrocarbyl-S-hydrocarbylene, hydrocarbyl-NH-hydrocarbylene,hydrocarbyl-amide-hydrocarbylene, N-(hydrocarbyl)hydrocarbylene,N,N-di(hydrocarbyl)hydrocarbylene, hydrocarbylacyl-hydrocarbylene,heterocyclylhydrocarbyl wherein the heteroatom(s) are selected fromoxygen, nitrogen, sulfur and phosphorus, substitutedheterocyclylhydrocarbyl wherein the heteroatom(s) are selected fromoxygen, nitrogen, sulfur and phosphorus and the substituents areselected from hydrocarbyl, hydrocarbyl-O-hydrocarbylene,hydrocarbyl-NH-hydrocarbylene, hydrocarbyl-S-hydrocarbylene,N-(hydrocarbyl)hydrocarbylene, N,N-di(hydrocarbyl)hydrocarbylene andhydrocarbylacyl-hydrocarbylene, as well as derivatives of any of theforegoing wherein one or more hydrogens is replaced with an equal numberof fluorides.
 27. A compound according to claim 25 wherein T³ has theformula —G(R²)—, G is C₁₋₆ alkylene having a single R² substituent, andR² is selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl-fusedcycloalkyl, cycloalkenyl, aryl, aralkyl, aryl-substituted alkenyl oralkynyl, cycloalkyl-substituted alkyl, cycloalkenyl-substitutedcycloalkyl, biaryl, alkoxy, alkenoxy, alkynoxy, aralkoxy,aryl-substituted alkenoxy or alkynoxy, alkylamino, alkenylamino oralkynylamino, aryl-substituted alkylamino, aryl-substituted alkenylaminoor alkynylamino, aryloxy, arylamino, N-alkylurea-substituted alkyl,N-arylurea-substituted alkyl, alkylcarbonylamino-substituted alkyl,aminocarbonyl-substituted alkyl, heterocyclyl, heterocyclyl-substitutedalkyl, heterocyclyl-substituted amino, carboxyalkyl substituted aralkyl,oxocarbocyclyl-fused aryl and heterocyclylalkyl; cycloalkenyl,aryl-substituted alkyl and, aralkyl, hydroxy-substituted alkyl,alkoxy-substituted alkyl, aralkoxy-substituted alkyl, alkoxy-substitutedalkyl, aralkoxy-substituted alkyl, amino-substituted alkyl,(aryl-substituted alkyloxycarbonylamino)-substituted alkyl,thiol-substituted alkyl, alkylsulfonyl-substituted alkyl,(hydroxy-substituted alkylthio)-substituted alkyl,thioalkoxy-substituted alkyl, hydrocarbylacylamino-substituted alkyl,heterocyclylacylamino-substituted alkyl,hydrocarbyl-substituted-heterocyclylacylamino-substituted alkyl,alkylsulfonylamino-substituted alkyl, arylsulfonylamino-substitutedalkyl, morpholino-alkyl, thiomorpholino-alkyl, morpholinocarbonyl-substituted alkyl, thiomorpholinocarbonyl-substituted alkyl,[N-(alkyl, alkenyl or alkynyl)- or N,N-[dialkyl, dialkenyl, dialkynyl or(alkyl, alkenyl)-amino]carbonyl-substituted alkyl,heterocyclylaminocarbonyl, heterocylylalkyleneaminocarbonyl,heterocyclylaminocarbonyl-substituted alkyl,heterocylylalkyleneaminocarbonyl-substituted alkyl,N,N-[dialkyl]alkyleneaminocarbonyl,N,N-[dialkyl]alkyleneaminocarbonyl-substituted alkyl, alkyl-substitutedheterocyclylcarbonyl, alkyl-substituted heterocyclylcarbonyl-alkyl,carboxyl-substituted alkyl, dialkylamino-substituted acylaminoalkyl andamino acid side chains selected from arginine, asparagine, glutamine,S-methyl cysteine, methionine and corresponding sulfoxide and sulfonederivatives thereof, glycine, leucine, isoleucine, allo-isoleucine,tert-leucine, norleucine, phenylalanine, tyrosine, tryptophan, proline,alanine, ornithine, histidine, glutamine, valine, threonine, serine,aspartic acid, beta-cyanoalanine, and allothreonine; alynyl,heterocyclylcarbonyl, aminocarbonyl, amido, mono- ordialkylaminocarbonyl, mono- or diarylaminocarbonyl,alkylarylaminocarbonyl, diarylaminocarbonyl, mono- ordiacylaminocarbonyl, aromatic or aliphatic acyl, alkyl optionallysubstituted by substituents selected from amino, carboxy, hydroxy,mercapto, mono- or dialkylamino, mono- or diarylamino, alkylarylamino,diarylamino, mono- or diacylamino, alkoxy, alkenoxy, aryloxy,thioalkoxy, thioalkenoxy, thioalkynoxy, thioaryloxy and heterocyclyl.28. A compound according to claim 25 having the formula:

wherein G is (CH₂)₁₋₆ wherein a hydrogen on one and only one of the CH₂groups of each G is replaced with —(CH₂)_(c)-Amide-T⁴; T² and T⁴ areorganic moieties of the formula C₁₋₂₅N₀₋₉S₀₋₃P₀₋₃H_(α)F_(β)I_(δ) whereinthe sum of α, β and δ is sufficient to satisfy the otherwise unsatisfiedvalencies of the C, N, O, S and P atoms; Amide is

 R¹ is hydrogen or C₁₋₁₀ alkyl; c is an integer ranging from 0 to 4; Xis defined according to claim 1; and n is an integer ranging from 1 to50 such that when n is greater than 1, G, c, Amide, R¹ and T⁴ areindependently selected.
 29. A compound according to claim 28 having theformula:

wherein T⁵ is an organic moiety of the formulaC₁₋₂₅N₀₋₉O₀₋₉S₀₋₃P₀₋₃H_(α)F_(β)I_(δ) wherein the sum of α, β and δ issufficient to satisfy the otherwise unsatisfied valencies of the C, N,O, S and P atoms; and T⁵ includes a tertiary or quaternary amine or anorganic acid; and m is an integer ranging from 0-49.
 30. A compoundaccording to claim 28 having the formula:

wherein T⁵ is an organic moiety of the formulaC₁₋₂₅N₀₋₉O₀₋₉S₀₋₃P₀₋₃H_(α)F_(β)I_(δ) wherein the sum of α, β and δ issufficient to satisfy the otherwise unsatisfied valencies of the C, N,O, S and P atoms; and T⁵ includes a tertiary or quaternary amine or anorganic acid; and m is an integer ranging from 0-49.
 31. A compoundaccording to any one of claims 29 and 30 wherein -Amide-T⁵ is selectedfrom:


32. A compound according to any of claims 29 and 30 wherein -Amide-T⁵ isselected from:


33. A compound according to any one of claims 25-29 wherein T² has thestructure which results when one of the following organic acids iscondensed with an amine group to form T²—C(═O)—N(R¹)—: Formic acid,Acetic acid, Propiolic acid, Propionic acid, Fluoroacetic acid,2-Butynoic acid, Cyclopropanecarboxylic acid, Butyric acid,Methoxyacetic acid, Difluoroacetic acid, 4-Pentynoic acid,Cyclobutanecarboxylic acid, 3,3-Dimethylacrylic acid, Valeric acid,N,N-Dimethylglycine, N-Formyl-Gly-OH, Ethoxyacetic acid,(Methylthio)acetic acid, Pyrrole-2-carboxylic acid, 3-Furoic acid,Isoxazole-5-carboxylic acid, trans-3-Hexenoic acid, Trifluoroaceticacid, Hexanoic acid, Ac-Gly-OH, 2-Hydroxy-2-methylbutyric acid, Benzoicacid, Nicotinic acid, 2-Pyrazinecarboxylic acid,1-Methyl-2-pyrrolecarboxylic acid, 2-Cyclopentene-1-acetic acid,Cyclopentylacetic acid, (S)-(−)-2-Pyrrolidone-5-carboxylic acid,N-Methyl-L-proline, Heptanoic acid, Ac-b-Ala-OH,2-Ethyl-2-hydroxybutyric acid, 2-(2-Methoxyethoxy)acetic acid, p-Toluicacid, 6-Methylnicotinic acid, 5-Methyl-2-pyrazinecarboxylic acid,2,5-Dimethylpyrrole-3-carboxylic acid, 4-Fluorobenzoic acid,3,5-Dimethylisoxazole-4-carboxylic acid, 3-Cyclopentylpropionic acid,Octanoic acid, N,N-Dimethylsuccinamic acid, Phenylpropiolic acid,Cinnamic acid, 4-Ethylbenzoic acid, p-Anisic acid,1,2,5-Trimethylpyrrole-3-carboxylic acid, 3-Fluoro-4-methylbenzoic acid,Ac-DL-Propargylglycine, 3-(Trifluoromethyl)butyric acid,1-Piperidinepropionic acid, N-Acetylproline, 3,5-Difluorobenzoic acid,Ac-L-Val-OH, Indole-2-carboxylic acid, 2-Benzofurancarboxylic acid,Benzotriazole-5-carboxylic acid, 4-n-Propylbenzoic acid,3-Dimethylaminobenzoic acid, 4-Ethoxybenzoic acid, 4-(Methylthio)benzoicacid, N-(2-Furoyl)glycine, 2-(Methylthio)nicotinic acid,3-Fluoro-4-methoxybenzoic acid, Tfa-Gly-OH, 2-Napthoic acid, Quinaldicacid, Ac-L-Ile-OH, 3-Methylindene-2-carboxylic acid,2-Quinoxalinecarboxylic acid, 1-Methylindole-2-carboxylic acid,2,3,6-Trifluorobenzoic acid, N-Formyl-L-Met-OH,2-[2-(2-Methoxyethoxy)ethoxy]acetic acid, 4-n-Butylbenzoic acid,N-Benzoylglycine, 5-Fluoroindole-2-carboxylic acid, 4-n-Propoxybenzoicacid, 4-Acetyl-3,5-dimethyl-2-pyrrolecarboxylic acid,3,5-Dimethoxybenzoic acid, 2,6-Dimethoxynicotinic acid,Cyclohexanepentanoic acid, 2-Naphthylacetic acid,4-(1H-Pyrrol-1-yl)benzoic acid, Indole-3-propionic acid,m-Trifluoromethylbenzoic acid, 5-Methoxyindole-2-carboxylic acid,4-Pentylbenzoic acid, Bz-b-Ala-OH, 4-Diethylaminobenzoic acid,4-n-Butoxybenzoic acid, 3-Methyl-5-CF3-isoxazole-4-carboxylic acid,(3,4-Dimethoxyphenyl)acetic acid, 4-Biphenylcarboxylic acid,Pivaloyl-Pro-OH, Octanoyl-Gly-OH, (2-Naphthoxy)acetic acid,Indole-3-butyric acid, 4-(Trifluoromethyl)phenylacetic acid,5-Methoxyindole-3-acetic acid, 4-(Trifluoromethoxy)benzoic acid,Ac-L-Phe-OH, 4-Pentyloxybenzoic acid, Z-Gly-OH,4-Carboxy-N-(fur-2-ylmethyl)pyrrolidin-2-one, 3,4-Diethoxybenzoic acid,2,4-Dimethyl-5-CO₂Et-pyrrole-3-carboxylic acid,N-(2-Fluorophenyl)succinamic acid, 3,4,5-Trimethoxybenzoic acid,N-Phenylanthranilic acid, 3-Phenoxybenzoic acid, Nonanoyl-Gly-OH,2-Phenoxypyridine-3-carboxylic acid,2,5-Dimethyl-1-phenylpyrrole-3-carboxylic acid,trans-4-(Trifluoromethyl)cinnamic acid,(5-Methyl-2-phenyloxazol-4-yl)acetic acid, 4-(2-Cyclohexenyloxy)benzoicacid, 5-Methoxy-2-methylindole-3-acetic acid, trans-4-Cotininecarboxylicacid, Bz-5-Aminovaleric acid, 4-Hexyloxybenzoic acid,N-(3-Methoxyphenyl)succinamic acid, Z-Sar-OH,4-(3,4-Dimethoxyphenyl)butyric acid, Ac-o-Fluoro-DL-Phe-OH,N-(4-Fluorophenyl)glutaramic acid, 4′-Ethyl-4-biphenylcarboxylic acid,1,2,3,4-Tetrahydroacridinecarboxylic acid, 3-Phenoxyphenylacetic acid,N-(2,4-Difluorophenyl)succinamic acid, N-Decanoyl-Gly-OH,(+)-6-Methoxy-a-methyl-2-naphthaleneacetic acid,3-(Trifluoromethoxy)cinnamic acid, N-Formyl-DL-Trp-OH,(R)-(+)-a-Methoxy-a-(trifluoromethyl)phenylacetic acid, Bz-DL-Leu-OH,4-(Trifluoromethoxy)phenoxyacetic acid, 4-Heptyloxybenzoic acid,2,3,4-Trimethoxycinnamic acid, 2,6-Dimethoxybenzoyl-Gly-OH,3-(3,4,5-Trimethoxyphenyl)propionic acid,2,3,4,5,6-Pentafluorophenoxyacetic acid,N-(2,4-Difluorophenyl)glutaramic acid, N-Undecanoyl-Gly-OH,2-(4-Fluorobenzoyl)benzoic acid, 5-Trifluoromethoxyindole-2-carboxylicacid, N-(2,4-Difluorophenyl)diglycolamic acid, Ac-L-Trp-OH,Tfa-L-Phenylglycine-OH, 3-Iodobenzoic acid,3-(4-n-Pentylbenzoyl)propionic acid, 2-Phenyl-4-quinolinecarboxylicacid, 4-Octyloxybenzoic acid, Bz-L-Met-OH, 3,4,5-Triethoxybenzoic acid,N-Lauroyl-Gly-OH, 3,5-Bis(trifluoromethyl)benzoic acid,Ac-5-Methyl-DL-Trp-OH, 2-lodophenylacetic acid, 3-Iodo-4-methylbenzoicacid, 3-(4-n-Hexylbenzoyl)propionic acid, N-Hexanoyl-L-Phe-OH,4-Nonyloxybenzoic acid, 4′-(Trifluoromethyl)-2-biphenylcarboxylic acid,Bz-L-Phe-OH, N-Tridecanoyl-Gly-OH, 3,5-Bis(trifluoromethyl)phenylaceticacid, 3-(4-n-Heptylbenzoyl)propionic acid, N-Hepytanoyl-L-Phe-OH,4-Decyloxybenzoic acid, N-(α,α,α-trifluoro-m-tolyl)anthranilic acid,Niflumic acid, 4-(2-Hydroxyhexafluoroisopropyl)benzoic acid,N-Myristoyl-Gly-OH, 3-(4-n-Octylbenzoyl)propionic acid,N-Octanoyl-L-Phe-OH, 4-Undecyloxybenzoic acid,3-(3,4,5-Trimethoxyphenyl)propionyl-Gly-OH, 8-Iodonaphthoic acid,N-Pentadecanoyl-Gly-OH, 4-Dodecyloxybenzoic acid, N-Palmitoyl-Gly-OH,and N-Stearoyl-Gly-OH.
 34. A composition comprising a plurality ofcompounds of the formula: T^(MS)—L—MOI wherein, T^(ms) is an organicgroup detectable by mass spectrometry, comprising carbon, at least oneof hydrogen and fluoride, and optional atoms selected from oxygen,nitrogen, sulfur, phosphorus and iodine; L is an organic group whichallows a T^(ms)-containing moiety to be cleaved from the remainder ofthe compound, wherein the T^(ms)-containing moiety comprises afunctional group which supports a single ionized charge state when thecompound is subjected to mass spectrometry and is selected from tertiaryamine, quaternary amine and organic acid; MOI is a nucleic acid fragmentwherein L is conjugated to the MOI at a location other than the 3′ endof the MOI; and wherein no two compounds have either the same T^(ms) orthe same MOI.
 35. A composition according to claim 34 wherein theplurality is greater than
 2. 36. A composition according to claim 34wherein the plurality is greater than
 4. 37. A composition according toclaim 34 wherein the nucleic acid fragment has a sequence complementaryto a portion of a vector, wherein the fragment is capable of primingnucleotide synthesis.
 38. A composition according to claim 34 whereinthe T^(ms) groups of members of the plurality differ by at least 2 amu.39. A composition according to claim 34 wherein the T^(ms) groups ofmembers of the plurality differ by at least 4 amu.
 40. A compositioncomprising water and a compound of claim
 15. 41. A composition accordingto claim 40 further comprising buffer, having a pH of about 5 to about9.
 42. A composition according to claim 40 further comprising an enzymeand one of dATP, dGTP, dCTP, and dTTP.
 43. A composition according toclaim 40 further comprising an enzyme and one of ddATP, ddGTP, ddCTP,and ddTTP.
 44. A composition comprising a plurality of sets ofcompounds, each set of compounds having the formula: T^(ms)—L—MOIwherein, T^(ms) is an organic group detectable by mass spectrometry,comprising carbon, at least one of hydrogen and fluoride, and optionalatoms selected from oxygen, nitrogen, sulfur, phosphorus and iodine; Lis an organic group which allows a T^(ms)-containing moiety to becleaved from the remainder of the compound, wherein theT^(ms)-containing moiety comprises a functional group which supports asingle ionized charge state when the compound is subjected to massspectrometry and is selected from tertiary amine, quaternary amine andorganic acid; MOI is a nucleic acid fragment wherein L is conjugated tothe MOI at a location other than the 3′ end of the MOI; wherein within aset, all members have the same T^(ms) group, and the MOI fragments havevariable lengths that terminate with the same dideoxynucleotide selectedfrom ddAMP, ddGMP, ddCMP and ddTMP; and wherein between sets, the T^(ms)groups differ by at least 2 amu.
 45. A composition according to claim 44wherein the plurality is at least
 3. 46. A composition according toclaim 44 wherein the plurality is at least
 5. 47. A compositioncomprising a first plurality of sets of compounds according to claim 44,and a second plurality of sets of compounds having the formulaT^(ms)—L—MOI wherein, T^(ms) is an organic group detectable by massspectrometry, comprising carbon, at least one of hydrogen and fluoride,and optional atoms selected from oxygen, nitrogen, sulfur, phosphorusand iodine; L is an organic group which allows a T^(ms)-containingmoiety to be cleaved from the remainder of the compound, wherein theT^(ms)-containing moiety comprises a functional group which supports asingle ionized charge state when the compound is subjected to massspectrometry and is selected from tertiary amine, quaternary amine andorganic acid; MOI is a nucleic acid fragment wherein L is conjugated tothe MOI at a location other than the 3′ end of the MOI; and wherein allmembers within the second plurality have an MOI sequence whichterminates with the same dideoxynucleotide selected from ddAMP, ddGMP,ddCMP and ddTMP; with the proviso that the dideoxynucleotide present inthe compounds of the first plurality is not the same dideoxynucleotidepresent in the compounds of the second plurality.
 48. A kit for DNAsequencing analysis comprising a plurality of container sets, eachcontainer set comprising at least five containers, wherein a firstcontainer contains a vector, a second, third, fourth and fifthcontainers contain compounds of the formula: T^(ms)—L—MOI wherein,T^(ms) is an organic group detectable by mass spectrometry, comprisingcarbon, at least one of hydrogen and fluoride, and optional atomsselected from oxygen, nitrogen, sulfur, phosphorus and iodine; L is anorganic group which allows a T^(ms)-containing moiety to be cleaved fromthe remainder of the compound, wherein the T^(ms)-containing moietycomprises a functional group which supports a single ionized chargestate when the compound is subjected to mass spectrometry and isselected from tertiary amine, quaternary amine and organic acid; and MOIis a nucleic acid fragment wherein L is conjugated to the MOI at alocation other than the 3′ end of the MOI; such that the MOI for thesecond, third, fourth and fifth containers is identical andcomplementary to a portion of the vector within the set of containers,and the T^(ms) group within each container is different from the otherT^(ms) groups in the kit.
 49. A kit according to claim 48 wherein theplurality is at least
 3. 50. A kit according to claim 48 wherein theplurality is at least
 5. 51. An apparatus for determining the sequenceof a nucleic acid molecule in a sample, the sample including taggednucleic acid fragments having nucleic acid fragments and tags attachedto the nucleic acid fragments, comprising a separation apparatus thatseparates tagged nucleic acid fragments, a cleavage apparatus thatreceives separated tagged cleaves nucleic acid fragments and the tagsfrom the nucleic acid fragments, each tag being correlative with aparticular nucleotide of the nucleic acid fragment and detectable byelectrochemical detection, and an apparatus for electrochemicaldetection that receives and detects electrochemical signatures of thetags.
 52. The apparatus according to claim 51 further including a dataprocessor that correlates the electrochemical signature of a tag to aparticular nucleotide and to a specific sample.
 53. The apparatusaccording to claim 51 wherein the apparatus for electrochemicaldetection is an apparatus for potentiostatic amperometry.
 54. Theapparatus according to claim 52 wherein the data processor is capable ofcorrelating the electrochemical signatures of five or more possible tagsto a particular nucleotide and to five or more specific samples.
 55. Theapparatus according to claim 52 wherein the data processor is capable ofcorrelating the electrochemical signatures of sixteen or more possibletags to a particular nucleotide and to sixteen or more specific samples.56. An apparatus for determining the sequence of a nucleic acid moleculein a sample, the sample including tagged nucleic acid fragments havingnucleic acid fragments and tags attached to the nucleic acid fragments,comprising a separation apparatus that separates tagged nucleic acidfragments, a cleavage apparatus that receives separated tagged nucleicacid fragments and cleaves from the nucleic acid fragments, each tagbeing correlative with a particular nucleotide of the nucleic acidfragment and detectable by mass spectrometry, a mass spectrometer thatreceives the tags and detects a mass of a tag, and a data processor thatcorrelates the mass of a tag to a particular nucleotide and to aspecific sample.
 57. The apparatus according to claim 56 wherein thedata processor is capable of correlating the masses of five or morepossible tags to a particular nucleotide and to five or more specificsamples.
 58. The apparatus according to claim 56 wherein the dataprocessor is capable of correlating the masses of sixteen or morepossible tags to a particular nucleotide and to sixteen or more specificsamples.